Human diacylglycerol acyltransferase 2 (DGAT2) family members and uses therefor

ABSTRACT

The present invention relates to compositions and methods for the diagnosis and treatment of obesity and related metabolic disorders. The invention provides isolated nucleic acids molecules, designated DGAT2 family member nucleic acid molecules, which encode diacylglycerol acyltransferase family members. The invention also provides recombinant expression vectors containing DGAT2 family member nucleic acid molecules, host cells into which the expression vectors have been introduced, and nonhuman transgenic animals in which a DGAT2 family member gene has been introduced or disrupted. The invention still further provides isolated DGAT2 family member proteins, fusion proteins, antigenic peptides and anti-DGAT2 family member antibodies. Methods of use of the provided DGAT2 family member compositions for screening, diagnostic and therapeutic methods in connection with obesity disorders are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.11/296,615, filed Dec. 7, 2005 (pending), which is a divisional of U.S.patent application Ser. No. 10/324,618, filed Dec. 19, 2002 (abandoned),which claims the benefit of U.S. Provisional Application No. 60/341,947,filed Dec. 19, 2001 (abandoned), and U.S. Provisional Application No.60/411,859, filed Sep. 19, 2002 (abandoned). Each of these applicationsis hereby incorporated in its entirety by reference herein.

BACKGROUND OF THE INVENTION

Obesity, the most prevalent of body weight disorders, is the mostimportant nutritional disorder in the western world, with estimates ofits prevalence ranging from 30% to 50% within the middle-agedpopulation. Obesity, defined as an excess of body fat relative to leanbody mass, also contributes to other diseases. For example, thisdisorder is responsible for increased incidence of diseases such ascoronary artery disease, hypertension, stroke, diabetes, hyperlipidemia,and some cancers (See, e.g., Nishina, P. M. et al., 1994, Metab. 43:554-558; Grundy, S. M. & Barnett, J. P., 1990, Dis. Mon. 36: 641-731).Obesity is not merely a behavioral problem, i.e., the result ofvoluntary hyperphagia. Rather, the differential body compositionobserved between obese and normal subjects results from differences inboth metabolism and neurologic/metabolic interactions. These differencesseem to be, to some extent, due to differences in gene expression,and/or level of gene products or activity. The nature, however, of thegenetic factors which control body composition are unknown, and attemptsto identify molecules involved in such control have generally beenempiric, and the parameters of body composition and/or substrate fluxhave not yet been identified (Friedman, J. M. et al., 1991, MammalianGene 1:130-144). The epidemiology of obesity strongly shows that thedisorder exhibits inherited characteristics (Stunkard, 1990, N. Eng. J.Med. 322:1483). Moll et al., have reported that, in many populations,obesity seems to be controlled by a few genetic loci (Moll et al. 1991,Am. J. Hum. Gen. 49:1243). In addition, human twin studies stronglysuggest a substantial genetic basis in the control of body weight, withestimates of heritability of 80-90% (Simopoulos, A. P. & Childs B.,eds., 1989, in “Genetic Variation and Nutrition in Obesity”, WorldReview of Nutrition and Diabetes 63, S. Karger, Basel, Switzerland;Borjeson, M., 1976, Acta. Paediatr. Scand. 65:279-287).

In other studies, non-obese persons who deliberately attempted to gainweight by systematically over-eating were found to be more resistant tosuch weight gain and able to maintain an elevated weight only by veryhigh caloric intake. In contrast, spontaneously obese individuals areable to maintain their status with normal or only moderately elevatedcaloric intake. Studies of the genetics of human obesity, and of animalmodels of obesity demonstrate that obesity results from complexdefective regulation of both food intake, food induced energyexpenditure, and of the balance between lipid and lean body anabolism.

It has now been established that the maintenance of body weight, satietyand energy expenditure is a complex process, regulated at variouslevels, including external and hypothalmic control of satiety,neuroendocrine and sympathetic nervous system control of metabolicprocesses, as well as enzymatic and transcriptional controls ofutilization of glucose, and adipogenesis (Kahn, 2000, Nature Genetics25: 6; and Palou, et al., 2000, Eur. J. Nutr. 39: 127).

It is estimated that approximately 40% of calories in the western dietare from fat. Thus, blocking absorption of a fraction of such fat wouldlead to weight loss. The pathways involved in fatty acid absorption inthe small intestine are fairly well understood. Fatty acids areliberated from triglycerides in the lumen of the small intestine throughthe action of pancreatic lipase. Free fatty acids then cross the plasmamembrane of the enterocytes, a transport mechanism probably utilizingFATP4, and, once in the enterocyte, are re-esterified intotriacylglycerols, the major form of energy stored in adipose tissue,which are packaged into chylomicrons prior to absorption.

Although production of diacylglycerol can be accomplished throughvarious mechanisms, the final rate-limiting step in biosynthesis oftriaclyglycerol is accomplished via the enzyme diacyl glycerolacyltransferase (DGAT). Although it has been known that DGAT activity isincreased in obese rodents, DGAT1 deficient mice are resistant to highfat-diet induced obesity and have increased energy expenditure (Smith,2000, Nature Genetics 25: 87). Until recently when a second DGAT enzyme(DGAT2) was identified, it was believed a single enzyme was responsiblefor synthesis of triacylglycerol (Cases et al. 2001 J. Biol. Chem. 276:38870). An understanding of regulation and maintenance of this ratelimiting step of triglyceride can provide insight into the regulation ofproduction and maintenance of energy stores and fat, and assist in thedevelopment of treatment for obesity and related disorders involvingproduction of triacylglycerols.

Given the importance of understanding body weight homeostasis and,further, given the severity and prevalence of disorders, includingobesity, which affect body weight and body composition, there exists agreat need for the systematic identification of genes and regulation ofgenes involved in these complex processes and disorders. Suchidentification will provide rationales and facilitate development ofspecific compounds acting via modulation of metabolic activity for usein the treatment of obesity and related disorders.

DESCRIPTION OF THE INVENTION

The present invention is based, in part, on the discovery of novel humandiacylglycerol acyltransferase 2 (DGAT2) family members, referred toherein as “60489,” “112041,” and “112037.” The nucleotide sequence ofcDNAs encoding 60489, 112041 and 112037 are shown in SEQ ID NO:7, SEQ IDNO:19, and SEQ ID NO:61 respectively; the amino acid sequences of 60489,112041, and 112037 polypeptides are shown in SEQ ID NO:8, SEQ ID NO:20,and SEQ ID NO:62.

Additionally, the invention is based on the discovery of novelexpression and regulation of human diacylglycerol acyltransferase 2(DGAT2) family members referred to herein as “58765,” “58765short,”“86606,” “112023,” “112024,” and “hDC2.” The nucleotide sequence of acDNA encoding 58765 is shown in SEQ ID NO:1, and the amino acid sequenceof a 58765 polypeptide is shown in SEQ ID NO:2. The nucleotide sequenceof a cDNA encoding 58765short is shown in SEQ ID NO:3, and the aminoacid sequence of a 58765 short polypeptide is shown in SEQ ID NO:4. Thenucleotide sequence of a cDNA encoding 86606 is shown in SEQ ID NO:9,and the amino acid sequence of a 86606 polypeptide is shown in SEQ IDNO:10. The nucleotide sequence of a cDNA encoding 112023 is shown in SEQID NO:13, and the amino acid sequence of a 112023 polypeptide is shownin SEQ ID NO:14. The nucleotide sequence of a cDNA encoding 112024 isshown in SEQ ID NO:17, and the amino acid sequence of a 112024polypeptide is shown in SEQ ID NO:18. The nucleotide sequence of a cDNAencoding hDC2 is shown in SEQ ID NO:21, and the amino acid sequence of ahDC2 polypeptide is shown in SEQ ID NO:22. Further, the presentinvention provides murine gene sequences were also identified which arerelated to DGAT2 sequences. The murine DGAT2 orthologue sequence(m86606) is depicted in SEQ ID NO:11, and the amino acid sequence of am86606 polypeptide is shown in SEQ ID NO:12. The murine DGAT2 familymember sequence m58765 sequence is shown in SEQ ID NO:5, and the aminoacid sequence of a m58765 polypeptide is shown in SEQ ID NO:6. The DGAT2family member nucleotide sequence of ml 12023 is shown in SEQ ID NO:15,and the amino acid sequence of a ml 12023 polypeptide is shown in SEQ IDNO:16. The DGAT2 family member nucleotide sequence of mDC2 is shown inSEQ ID NO:23, and the amino acid sequence of a mDC2 polypeptide is shownin SEQ ID NO:24.

Accordingly, in one aspect, the invention features nucleic acidmolecules which encode a DGAT2 family member protein or polypeptide, ora fragment thereof, e.g., a biologically active portion of the DGAT2family member protein. In a preferred embodiment, the isolated nucleicacid molecule encodes a polypeptide having the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, or SEQ ID NO:62. In other embodiments, theinvention provides an isolated DGAT2 family member nucleic acid moleculehaving the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or SEQ IDNO:61. In still other embodiments, the invention provides nucleic acidmolecules that are substantially identical (e.g., naturally occurringallelic variants) to the nucleotide sequence shown in SEQ ID NO:1, SEQID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, or SEQ ID NO:61. In other embodiments, the invention provides anucleic acid molecule which hybridizes under stringent hybridizationconditions to a nucleic acid molecule comprising the nucleotide sequenceof SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ IDNO:21, SEQ ID NO:23, or SEQ ID NO:61, wherein the nucleic acid encodes afull length DGAT2 family member protein or an active fragment thereof.

In a related aspect, the invention further provides nucleic acidconstructs which include a DGAT2 family member nucleic acid moleculedescribed herein. In certain embodiments, the nucleic acid molecules ofthe invention are operatively linked to native or heterologousregulatory sequences. Also included, are vectors and host cellscontaining the DGAT2 family member nucleic acid molecules of theinvention e.g., vectors and host cells suitable for producing DGAT2family member nucleic acid molecules and polypeptides.

In another related aspect, the invention provides nucleic acid of DGAT2family member-encoding nucleic acids. The fragments of the invention canbe suitable as primers or hybridization probes for the detection ofDGAT2 family member encoding nucleic acids.

In still another related aspect, isolated nucleic acid molecules thatare antisense to a DGAT2 family member encoding nucleic acid moleculeare provided.

In another aspect, the invention features, DGAT2 family memberpolypeptides, and biologically active or antigenic fragments thereofthat are useful, e.g., as reagents or targets in assays applicable totreatment and diagnosis of DGAT2 family member-mediated or -relateddisorders. In another embodiment, the invention provides DGAT2 familymember polypeptides having a DGAT2 family member activity. Preferredpolypeptides are DGAT2 family member proteins including at least oneacyltransferase domain, and/or plsC domain, and, preferably, having aDGAT2 family member activity, e.g., a DGAT2 family member activity asdescribed herein.

In other embodiments, the invention provides DGAT2 family memberpolypeptides, e.g., a DGAT2 family member polypeptide having the aminoacid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:62; anamino acid sequence that is substantially identical to the amino acidsequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18,SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:62; or an aminoacid sequence encoded by a nucleic acid molecule having a nucleotidesequence which hybridizes under stringent hybridization conditions to anucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23, or SEQ ID NO:61, wherein the nucleic acid encodes a full lengthDGAT2 family member protein or an active fragment thereof. In a relatedaspect, the invention further provides nucleic acid constructs whichinclude a DGAT2 family member nucleic acid molecule described herein.

In a related aspect, the invention provides DGAT2 family memberpolypeptides or fragments operatively linked to non-DGAT2 family memberpolypeptides to form fusion proteins.

In another aspect, the invention features antibodies and antigen-bindingfragments thereof, that react with, or more preferably specifically bindDGAT2 family member polypeptides.

The present invention is based, at least in part, on the discovery thatDGAT2 family member molecules are expressed at increased levels inadipose, liver, small intestine, colon, and kidney tissues, (seeExamples 3-7 and Tables 1-8 described herein). DGAT2 family membermolecules were further found to be upregulated during adipocytedifferentiation, and downregulated during exposure to starvationconditions or mice fed high fat diets (i.e., under conditions thataffect adipocyte metabolism) as well as in genetic models of obesity(see Example 3 and Tables 3-8).

Accordingly, the present invention provides methods for the diagnosisand treatment of metabolic and related disorders including but notlimited to obesity, hyperlipidemia and other lipid disorders anddiabetes.

In one aspect, the invention provides methods of screening for compoundsthat modulate the expression or activity of the DGAT2 family memberpolypeptides or nucleic acids. The method includes contacting a sampleexpressing a DGAT2 family member nucleic acid or polypeptide with a testcompound and assaying the ability of the test compound to modulate theexpression of a DGAT2 family member nucleic acid or the activity of aDGAT2 family member polypeptide.

In one embodiment, the invention provides methods for identifying acompound capable of treating a metabolic disorder, e.g., obesity,hyperlipidemia, and diabetes. The method includes assaying the abilityof the compound to modulate DGAT2 family member nucleic acid expressionor DGAT2 family member polypeptide activity. In one embodiment, theability of the compound to modulate nucleic acid expression or DGAT2family member polypeptide activity is determined by detecting modulationof lipogenesis.

In another embodiment, the ability of the compound to modulate nucleicacid expression or DGAT2 family member polypeptide activity isdetermined by detecting modulation of triglyceride biosynthesis. Instill another embodiment, the ability of the compound to modulatenucleic acid expression or DGAT2 family member polypeptide activity isdetermined by detecting modulation of hyperplastic growth. In yetanother embodiment, the ability of the compound to modulate nucleic acidexpression or DGAT2 family member polypeptide activity is determined bydetecting modulation of hypertrophic growth.

In another aspect, the invention provides methods for identifying acompound capable of modulating an adipocyte activity, e.g., hyperplasticgrowth, hypertrophic growth, or lipogenesis. The method includescontacting an adipocyte expressing a DGAT2 family member nucleic acid orpolypeptide with a test compound and assaying the ability of the testcompound to modulate the expression of a DGAT2 family member nucleicacid or the activity of a DGAT2 family member polypeptide.

In still another aspect, the invention provides methods for determiningacyltransferase activity of a polypeptide. Such methods includecombining a sample comprising an acyltransferase polypeptide with afatty acyl coA substrate and a acylglyceride substrate under conditionssuitable to carry out enzyme activity, and determining the amount ofacylglycerol product formed, wherein product formation is adetermination of acylglycerol-acyltransferase activity. In certainaspects, one substrate can be biotinylated and the other substrate canbe radiolabeled (e.g., radiolabeled acylglyceride and biotinylated fattyacyl coA). Product formation can be determined using biotin capture andradiometric determination (eg, SPA (scintillation proximity assay))assays. Provided acyltransferase activity methods can be used to detectany acyltransferase activity (e.g., monoacylglycerol acyltransferase,diacylglycerol acyltransferase). In particular embodiments,acyltransferase activity methods can be used to determine enzymeactivity of the DGAT2 family members provided herein.

Yet another aspect includes methods for identifying compounds whichmodulate acyltransferase activity. The provided methods include thedescribed methods for determining acyltransferase activity, withadditional steps including contacting the sample comprising anacyltransferase polypeptide and fatty acyl coA and acylglyceridesubstrates with one or more test compounds. Test compounds can be addedto the sample at any time before, during, or after combining thecomposition comprising acyltransferase and substrates. Enzyme activityis determined by measuring product formation, wherein a change in theamount of acyltransferase activity in the presence of test compoundidentifies a compound which modulates acyltransferase activity.

In another aspect, the invention provides methods for modulating anadipocyte activity, e.g., hyperplastic growth, hypertrophic growth, orlipogenesis. The method includes contacting an adipocyte with a DGAT2family member modulator, for example, an anti-DGAT2 family memberantibody, a DGAT2 family member polypeptide comprising the amino acidsequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:62, or a fragmentthereof, a DGAT2 family member polypeptide comprising an amino acidsequence which is at least 90 percent identical to the amino acidsequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:62, an isolatednaturally occurring allelic variant of a polypeptide consisting of theamino acid sequence of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:62, asmall molecule, an antisense DGAT2 family member nucleic acid molecule,a nucleic acid molecule of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:61, or afragment thereof, or a ribozyme.

In still another aspect, the invention provides a process for modulatingDGAT2 family member polypeptide or nucleic acid expression or activity,e.g. using the screened compounds. In certain embodiments, the methodsinvolve treatment of conditions related to aberrant activity orexpression of the DGAT2 family member polypeptides or nucleic acids,such as conditions involving aberrant or deficient triglyceridebiosynthesis (e.g., obesity, lipid disorders).

The invention also provides assays for determining the activity of orthe presence or absence of DGAT2 family member polypeptides or nucleicacid molecules in a biological sample, including for disease diagnosis.In one aspect, provided are assays for determining the presence orabsence of a genetic alteration in a DGAT2 family member polypeptide ornucleic acid molecule, including for disease diagnosis.

In one embodiment, methods include identifying a nucleic acid associatedwith a metabolic disorder, e.g., obesity, hyperlipidemia, and diabetes.

In yet another aspect, the invention features a method for identifying asubject having an obesity disorder characterized by aberrant DGAT2family member polypeptide activity or aberrant DGAT2 family membernucleic acid expression. The method includes contacting a sampleobtained from the subject and expressing a DGAT2 family member nucleicacid or polypeptide with a test compound and assaying the ability of thetest compound to modulate the expression of a DGAT2 family membernucleic acid or the activity of a DGAT2 family member polypeptide.

In yet another aspect, the invention features a method for treating asubject having a metabolic disorder, e.g., obesity, diabetes,hyperlipidemia, characterized by aberrant DGAT2 family memberpolypeptide activity or aberrant DGAT2 family member nucleic acidexpression. The method includes administering to the subject a DGAT2family member modulator, e.g., in a pharmaceutically acceptableformulation or by using a gene therapy vector. Embodiments of thisaspect of the invention include the DGAT2 family member modulator beingany of an organic small molecule, an anti-DGAT2 family member antibody,a DGAT2 family member polypeptide comprising the amino acid sequence ofSEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ IDNO:22, SEQ ID NO:24, or SEQ ID NO:62, or a fragment thereof, a DGAT2family member polypeptide comprising an amino acid sequence which is atleast 90 percent identical to the amino acid sequence of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, or SEQ ID NO:62, an isolated naturally occurring allelic variantof a polypeptide consisting of the amino acid sequence of SEQ ID NO:2,SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ IDNO:24, or SEQ ID NO:62, an antisense DGAT2 family member nucleic acidmolecule, a nucleic acid molecule of SEQ ID NO:1, SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or SEQ IDNO:61, or a fragment thereof, or a ribozyme.

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as “DGAT2 family member” nucleicacid and polypeptide molecules, which play a role in, or function in,catalyzing the final step in the re-esterification of fatty acids toproduce triglycerols, and/or play a role in production and regulation offat and energy stores in mammals. This metabolic pathway is described inLodish et al. (1995) Molecular Cell Biology (Scientific American BooksInc., New York, N.Y.) and Stryer Biochemistry, (W.H. Freeman, New York),the contents of which are incorporated herein by reference. In oneembodiment, the DGAT2 family member molecules modulate the activity ofone or more proteins involved in production of triacylglycerols, and/orproduction of fat stores e.g., adipose fat stores. In anotherembodiment, the DGAT2 family member molecules of the present inventionare capable of modulating the esterification state of fatty acidmolecules for the production of one or more molecules involved inadipose energy stores, as described in, for example, Lodish et al. andStryer, supra. Additionally, the DGAT2 family members of the inventionmay modulate triglyceride production and energy storage in tissues andcells including liver, small intestine, kidney, adipose, skeletalmuscle, pancreas, heart, spleen, brain, hypothalamus, lung, etc.

As used herein, the term “diacylglycerol acyltransferase”“acyl-CoA:diacylglycerol acyltransferase” or “DGAT” includes a protein,polypeptide, or other non-proteinaceous molecule that is capable ofmodulating the esterification state of diacylglycerol (DAG) molecules.DGATs play a role in biosynthetic pathways associated with production offat stores. For example, DGATs are involved in the regulation ofbiosynthesis of triacylglycerols. The enzyme reaction catalyzed byAcyl-CoA:diacylglycerol acyltransferases (DGATs) involves the couplingof an acyl-CoA (1) to a preformed diacylglycerol (2) producing oneequivalent of Coenzyme A (CoA) and triacylglycerol.

Novel DGAT Sequences

The present invention is based, at least in part, on the discovery ofnovel molecules, referred to herein as diacylglycerol acyltransferase 2(DGAT2) family member protein and nucleic acid molecules, that comprisea family of molecules having certain conserved structural and functionalfeatures. The term “family” when referring to the protein and nucleicacid molecules of the invention is intended to mean two or more proteinsor nucleic acid molecules having a common structural domain or motif andhaving sufficient amino acid or nucleotide sequence identity as definedherein. Such family members can be naturally or non-naturally occurringand can be from either the same or different species. For example, afamily can contain a first protein of human origin, as well as other,distinct proteins of human origin or alternatively, can containhomologues of non-human origin. Members of a family may also have commonfunctional characteristics.

One embodiment of the invention features diacylglycerol acyltransferase2 (DGAT2) family member nucleic acid molecules, preferably human DGAT2family member molecules, that were initially identified based on relatedsequence or protein domain characteristic of acyl glycerol phosphateacyltransferase family of proteins. Such sequences are referred to as“DGAT2 family member” sequences indicating that the genes share sequencesimilarity with diacylglycerol acyltransferase 2 gene. Specifically,novel human DGAT2 family member family members, 60489, 112041, and112037 are provided. They are highly expressed in small intestine,adipose, and liver where triglyceride synthesis occurs.

In addition, we have demonstrated tissue expression and regulation ofadditional human DGAT2 family member family members 86606, 58765,112023, 112024, hDC2, as well as murine orthologues m86606, m58765, ml12023, and mDC2. They are also highly expressed in tissues wheretriglyceride synthesis occurs, expression is regulated under conditionsthat change adipocyte metabolism both in vitro and in vivo. DGAT2 familymember family members are therefore a candidate target to identify smallmolecules for the treatment of obesity, diabetes, and/or lipid disordersin humans. It is conceivable that inhibition of these genes, eitherindividually or collectively, will lead to decreased triglyceridesynthesis and fat accumulation in vivo. Inhibitors, therefore, havepotentials for anti-fat absorption and can be used to treat obesity andits related disorders.

Human DGAT2 Family Members

The human DGAT2, (herein referred to as 86606) sequence is depicted inSEQ ID NO:9, which is approximately 2428 nucleotides long includinguntranslated regions, contains a predicted methionine-initiated codingsequence of about 1166 nucleotides (nucleotides 220-1386 of SEQ IDNO:9). The coding sequence encodes a 388 amino acid protein (SEQ IDNO:10). The molecule may have transmembrane segments from amino acids(aa) 70-93 and 100-116 as predicted by MEMSAT. Prosite program analysiswas used to predict various sites within the 86606 protein.N-glycosylation sites were predicted at aa 60-63, 173-176 and 228-231.Protein kinase C phosphorylation sites were predicted at aa 23-35,37-39, 116-118, 152-154, 182-184, and 255-257. Casein kinase IIphosphorylation sites were predicted at aa 62-65, 278-281, and 351-354.N-myristoylation sites were predicted at aa 10-15, 41-46, 84-89,120-125, 169-174, 229-234, 240-245, 318-323, and 378-383. An amidationsite was predicted at aa 120-123. The 86606 protein possesses a SMARTplsc-2 domain, from about aa 165 to about aa 281, as predicted by HMMer,Version 2.1.1. The plsc domain is believed to function in phospholipidbiosynthesis and is characteristic of proteins having glycerolphosphate,1-acylglycerolphosphate, or 2-acylglycerolphosphoethanolamineacyltransferase activities.

The human DGAT2 family member sequence 60489 (SEQ ID NO:7), which isapproximately 1255 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 1025nucleotides (nucleotides 170-1195 of SEQ ID NO:7) The coding sequenceencodes a 341 amino acid protein (SEQ ID NO:8). The molecule may havetransmembrane segments from amino acids (aa) 39-63, 109-127, and 271-291as predicted by MEMSAT. Prosite program analysis was used to predictvarious sites within the 60489 protein. N-glycosylation sites werepredicted at aa 126-129. Protein kinase C phosphorylation sites werepredicted at aa 12-14, and 255-257. Casein kinase II phosphorylationsites were predicted at aa 231-234, 304-307, and 317-320.N-myristoylation sites were predicted at aa 2-7, 73-78, 117-122,193-198, 271-276, and 331-336. An amidation site was predicted at aa73-76. The 60489 protein possesses a SMART plsc_(—)2 domain, from aboutaa 110 to about aa 234, as predicted by HMMer, Version 2.1.1. The plscdomain is believed to function in phospholipid biosynthesis and ischaracteristic of proteins having glycerolphosphate,1-acylglycerolphosphate, or 2-acylglycerolphosphoethanolamineacyltransferase activities.

The DGAT2 family member sequence 112041 (SEQ ID NO:19), which isapproximately 1716 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 1013nucleotides (nucleotides 101-1114 of SEQ ID NO:19) The coding sequenceencodes a 337 amino acid protein (SEQ ID NO:20). The molecule may havetransmembrane segments from amino acids (aa) 21-42 as predicted byMEMSAT. Prosite program analysis was used to predict various siteswithin the 112041 protein. An N-glycosylation site was predicted at aa75-78. Protein kinase C phosphorylation sites were predicted at aa97-99, 172-174, and 252-254. Casein kinase II phosphorylation sites werepredicted at aa 224-227, 235-238, and 248-251. N-myristoylation siteswere predicted at aa 66-71, 115-120, 175-180, 186-191, 258-263, and327-332. An amidation site was predicted at aa 66-69.

The human DGAT2 family member sequence 112037 (SEQ ID NO:61), is apartial sequence approximately 712 nucleotides long, contains apredicted coding sequence of about 711 nucleotides (nucleotides 2-712 ofSEQ ID NO:61) The coding sequence encodes a 236 amino acid protein (SEQID NO:62). The molecule may have transmembrane segments from amino acids(aa) 22-42 and 49-73, as predicted by MEMSAT. Prosite program analysiswas used to predict various sites within the 112037 protein. A Proteinkinase C phosphorylation sites was predicted at aa 4-6. A Casein kinaseII phosphorylation sites was predicted at aa 116-119. N-myristoylationsites were predicted at aa 8-13, 26-31, 68-73, and 84-89. An amidationsite was predicted at aa 156-159.

The DGAT2 family member sequence of 58765 identified two splice variantsequences including 58765 (SEQ ID NO:1), which is approximately 1005nucleotides long, encodes a 334 amino acid protein (SEQ ID NO:2). Themolecule may have dileucine motifs in the tail at about amino acids (aa)41-42, 48-49, 180-181, and 201-202, as predicted by PSORT. The moleculemay have transmembrane segments from amino acids (aa) 38-59 and 103-119as predicted by MEMSAT. Prosite program analysis was used to predictvarious sites within the 86606 protein. N-glycosylation sites werepredicted at aa 237-240. Protein kinase C phosphorylation sites werepredicted at aa 163-165. Casein kinase II phosphorylation sites werepredicted at aa 163-166, 225-228, and 297-300. N-myristoylation siteswere predicted at aa 116-121, 159-164, 178-183, and 187-192. The 58765protein possesses a SMART plsc_(—)2 domain, from about aa 111 to aboutaa 228, as predicted by HMMer, Version 2.1.1. The plsc domain isbelieved to function in phospholipid biosynthesis and is characteristicof proteins having glycerolphosphate, 1-acylglycerolphosphate, or2-acylglycerolphosphoethanolamine acyltransferase activities. Inaddition, the 58765 protein possess a PFAM acyltransferase domain, fromabout aa 104 to about aa 296, as predicted by HMMer, Version 2.1.1.

Additionally, 58765short (SEQ ID NO:3), which is approximately 855nucleotides long, encodes a 284 amino acid protein (SEQ ID NO:4). Themolecule may have transmembrane segments from amino acids (aa) 38-59 and103-119 as predicted by MEMSAT. Dileucine motifs may be present in thetail at aa 41-42, 48-49, 180-181, and 201-202, as predicted by PSORT.Prosite program analysis was used to predict various sites within the58765short protein. A cAMP and cGMP dependent protein kinasephosphorylation site was predicted at aa 277-280. Protein kinase Cphosphorylation sites were predicted at aa 163-165, 221-223, and258-260. Casein kinase II phosphorylation sites were predicted at aa163-166, and 244-247. N-myristoylation sites were predicted at aa116-121, 159-164, 178-183, 187-192, 227-232, and 238-243. An ATP/GTPbinding site motif was predicted at aa 217-224.

The DGAT2 family member sequence 112023 (SEQ ID NO:13), which isapproximately 1279 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 986nucleotides (nucleotides 42-1028 of SEQ ID NO:13) The coding sequenceencodes a 328 amino acid protein (SEQ ID NO:14. The molecule may havetransmembrane segments from about amino acids (aa) 13-29, 36-54, 98-116and 165-183 as predicted by MEMSAT. Dileucine motifs may be present inthe tail at aa 15-16, as predicted by PSORT. Prosite program analysiswas used to predict various sites within the 112023 protein. A proteinkinase C phosphorylation site was predicted at aa 322-324. A caseinkinase II phosphorylation site was predicted at aa 219-222.N-myristoylation sites were predicted at aa 62-67, 111-116, 172-177,181-186, 257-262, and 318-323. An amidation site was predicted at aa62-65.

The DGAT2 family member sequence 112024 (SEQ ID NO:17), which isapproximately 1720 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 1001nucleotides (nucleotides 1-1002 of SEQ ID NO:17) The coding sequenceencodes a 333 amino acid protein (SEQ ID NO:18). The molecule may havetransmembrane segments from about amino acids (aa) 37-58, and 130-150 aspredicted by MEMSAT. Dileucine motifs may be present in the tail at aa26-27, 90-91, 170-171, and 272-273, as predicted by PSORT. AnN-glycosylation sites was predicted at aa 204-207. A cAMP and cGMPdependent protein kinase phosphorylation site was predicted at aa 68-71.Protein kinase C phosphorylation sites were predicted at aa 5-7, and172-174. Casein kinase II phosphorylation sites were predicted at aa5-8, 11-14, and 165-168. N-myristoylation sites were predicted at aa186-191, 239-244, and 323-328. An amidation site was predicted at aa66-69. The 112024 protein possesses a SMART plsc_(—)2 domain, from aboutaa 118 to about aa 314, as predicted by HMMer, Version 2.1.1. The plscdomain is believed to function in phospholipid biosynthesis and ischaracteristic of proteins having glycerolphosphate,1-acylglycerolphosphate, or 2-acylglycerolphosphoethanolamineacyltransferase activities. The 112024 protein possesses a PFAMacyltransferase domain, from about aa 103 to about aa 227, as predictedby HMMer, Version 2.1.1.

The DGAT2 family member sequence hDC2 (SEQ ID NO:21), which isapproximately 1093 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 1004nucleotides (nucleotides 49-1053 of SEQ ID NO:21) The coding sequenceencodes a 334 amino acid protein (SEQ ID NO:22). The molecule may havetransmembrane segments from amino acids (aa) 19-43, 131-151 and 209-227as predicted by MEMSAT. Prosite program analysis was used to predictvarious sites within the hDC2 protein. N-glycosylation sites werepredicted at aa 76-79, 120-123, 124-127, and 179-182. A cAMP and cGMPdependent protein kinase phosphorylation site was predicted at aa 69-72.Protein kinase C phosphorylation sites were predicted at aa 164-166, and275-277. Casein kinase II phosphorylation sites were predicted at aa225-228, and 307-310. N-myristoylation sites were predicted at aa 67-72,116-121, 177-182, and 187-192. An amidation site was predicted at aa67-70.

In one embodiment, a DGAT2 family member molecule may include a signalsequence. As used herein, a “signal sequence” refers to a peptide ofabout 10-80 amino acid residues in length which occurs at the N-terminusof secretory and integral membrane proteins and which contains amajority of hydrophobic amino acid residues. For example, a signalsequence contains at least about 20-60 amino acid residues, preferablyabout 30-50 amino acid residues, more preferably about 37 amino acidresidues, and has at least about 40-70%, preferably about 50-65%, andmore preferably about 55-60% hydrophobic amino acid residues (e.g.,alanine, valine, leucine, isoleucine, phenylalanine, tyrosine,tryptophan, or proline). Such a “signal sequence”, also referred to inthe art as a “signal peptide”, serves to direct a protein containingsuch a sequence to a lipid bilayer. For example, in certain embodiments,a DGAT2 family member protein may contain a signal sequence of aboutamino acids 1-68 of SEQ ID NO:8, 1-65 of SEQ ID NO:20, 1-55 of SEQ IDNO:2, 1-55 of SEQ ID NO:4, 1-49 of SEQ ID NO:14, 1-58 of SEQ ID NO:18,or 1-63 of SEQ ID NO:22. The “signal sequence” is cleaved duringprocessing of the mature protein. In such embodiments, the mature DGAT2family member protein corresponds to amino acids 69-341 of SEQ ID NO:8,66-337 of SEQ ID NO:20, 56-334 of SEQ ID NO:2, 56-284 of SEQ ID NO:4,50-112023 of SEQ ID NO:14, 59-333 of SEQ ID NO:18, or 64-334 of SEQ IDNO:22.

Based on DGAT2 family member protein sequence, cellular localizationsignals can be identified by methods known to one of skill in the art(e.g., PSORT Prediction). Subcellular localization of a DGAT2 familymember, generated using PSORT Prediction software. Predictedtransmembrane domains may be identified by ORF analysis with MEMSAT.

For general information regarding PSORT, Prosite and PFAM identifiers,PS prefix and PF prefix domain identification numbers, refer toSonnhammer et al. (1997) Protein 28:405-420.

The DGAT2 family member protein contains a significant number ofstructural characteristics in common with members of the acyltransferasefamily. The term “family” when referring to the protein and nucleic acidmolecules of the invention means two or more proteins or nucleic acidmolecules having a common structural domain or motif and havingsufficient amino acid or nucleotide sequence homology as defined herein.Such family members can be naturally or non-naturally occurring and canbe from either the same or different species. For example, a family cancontain a first protein of human origin as well as other distinctproteins of human origin, or alternatively, can contain homologues ofnon-human origin, e.g., rat or mouse proteins. Members of a family canalso have common functional characteristics.

As used herein, the term “diacylglyceroltransferase” or “DGAT” refers toa family of proteins that preferably comprise a membrane boundacyltransferase enzyme. Members of the DGAT2 family also share certainconserved amino acid residues, some of which may be determined to becritical to acyltransferase function triglyceride biosynthesis. Forexample, alignment of the human DGAT2 family members is depicted inTable 1 below.

TABLE 1 Sequence alignment of human DGAT2 family members 112037 (SEQ IDNO: 62) .......... .......... .......... .......... .......... 60489(SEQ ID NO: 8) .......... .......... .......... .......... .......MGVDC2 (SEQ ID NO: 22) .......... .......... .......... .................... 112041 (SEQ ID NO: 20) .......... .......... .................... .......... 112024 (SEQ ID NO: 18) .......... .................... .......... .......... 112023 (SEQ ID NO: 16) .................... .......... .......... .......... 86606 (SEQ ID NO: 10)MKTLIAAYSG VLRGERQAEA DRSQRSHGGP ALSREGSGRW GTGSSILSAL 58765s (SEQ IDNO: 4) .......... .......... .......... .......... .......... 58765 (SEQID NO: 2) .......... .......... .......... .......... .......... 112037.......... .......... .......... .......... .......... 60489 ATTLQPPTTSKTLQKQHLEA VGAYQYVLTF LFMG.FFFSL LVFVLLFTSL DC2 ..MKVEFAPL NIQLARRLQTVAVLQWVLSF LTGP.MSIGI TVMLIIHM.Y 112041 .....MAFFS RLNLQEGLQT FFVLQWIPVYIFLGAIPILL IPYFLLFSKF 112024 .....MLLPS KKDLKTALDV FAVFQWSFSA LLITTTVIAVNLYLVVFTPY 112023 ......MAHS KQ..PSHFQS LMLLQWPLSY LAIFWILQPL FVYLL.FTSL86606 QDLFSVTWLN RSKVEKQLQV ISVLQWVLSF LVLGVACSAI LMYIF.CTDC 58765s...MVEFAPL FMPWERRLQT LAVLQFVFSF LALA.EICTV GFIALLFTRF 58765 ...MVEFAPLFMPWERRLQT LAVLQFVFSF LALA.EICTV GFIALLFTRF 112037 .......... .................... .......... ....LVKTAK 60489 WPFSVFYLVW LYVDWDTPNQ GGRRSEWIRNRAIWRQLRDY YPVKLVKTAE DC2 LFLYIPYLMW LYFDWHTPER GGRRSSWIKN WTLWKHFKDYFPIHLIKTQD 112041 WPLAVLSLAW LTYDWNTHSQ GGRRSAWVRN WTLWKYFRNY FPVKLVKTHD112024 WPVTVLILTW LAFDWKTPQR GGRRFTCVRH WRLWKHYSDY FPLKLLKTHD 112023WPLPVLYFAW LFLDWKTPER GGRRSAWVRN WCVWTHIRDY FPITILKTKD 86606 WLIAVLYFTWLVFDWNTPKK GGRRSQWVRN WAVWRYFRDY FPIQLVKTHN 58765s WLLTVLYAAW WYLDRDKPRQGGRHIQAIRC WTIWKYMKDY FPISLVKTAE 58765 WLLTVLYAAW WYLDRDKPRQ GGRHIQAIRCWTIWKYMKDY FPISLVKTAE 112037 LGTSWNYLFD FHPHRVLVVG AFANFCTEPT GCSCLFPKLPPHLLMLPCWF 60489 LPPDRNYVLG AHPHGIMCTG FLCNFSTESN GFSQLFPGLR PWLAVLAGLFDC2 LDPSHNYIFG FHPHGIMAVG AFGNFSVNYS DFKDLFPGFT SYLHVLPLWF 112041LSFKHNYIIA NHPHGILSFG VEINFATEAT GIARIFPSIT PFVGTLERIF 112024 ICPSRNYILVCHPHGLFAHG WFGHFATEAS GFSKIFPGIT PYILTLGAFF 112023 LSPEHNYLMG VHPHGLLTFGAFCNFCTEAT GFSKTFPGIT PHLATLSWFF 86606 LLTTRNYIFG YHPHGIMGLG AFCNFSTEATEVSKKFPGIR PYLATLAGNF 58765s LDPSRNYIAG FHPHGVLAVG AFANLCTEST GFSSIFPGIRPHLMMPTLWF 58765 LDPSRNYIAG FHPHGVLAVG AFANLCTEST GFSSIFPGIR PHLMMLTLWF112037 HLLFFQDYIM SGGLVSFVKA PLPQWWPGG. ...CP..GVG GPLQALEAKP 60489YLFVYRDYIM SFGLCFVSRQ SLDFILSQFQ LGQAVVIMVG GAHEALYSVP DC2 WCPVFREYVMSVGLVSVSKK SVSYMVSKEG GGNISVIVLG GAKESLDAHP 112041 WIPIVREYVM SMGVCPVSSSALKYLLTQKG SGNAVVIVVG GAAEALLCRP 112024 WMPFLREYVM STGACSVSRS SIDFLLTHKGTGNMVIVVIG GLAECRYSLP 112023 KIPFVREYLM AKGVCSVSQP AINYLLSHG. TGNLVGIVVGGVGEALQSVP 86606 EMPVLREYLM SGGICPVSRD TIDYLLSKNG SGNAIIIVVG GAAESLSSMP58765s RAPFFRDYIM SAGLVTSEKE SAAHILNRKG GGNLLGIIVG GAQEALDARP 58765RAPFFRDYIM SAGLVTSEKE SAAHILNRKG GGNLLGIIVG GAQEALDARP 112037 GQLSLPIRNQKRLVKSALEL .......... GENELFQQFP NPQSSWVQRT 60489 GEHCLTLQKR KGFVRLALRHGASLVPVYSF GENDIFRLKA FATGSWQHWC DC2 GKFTLFIRQR KGFVKIALTH GASLVPVVSFGEMELFKQTD NPEGSWIRTV 112041 GASTLFLKQR KGFVKMALQT GAYLVPSYSF GENEVFNQETFPEGTWLRLF 112024 GSSTLVLKNR SGFVRMALQH GVPLIPAYAF GETDLYDQHI FTPGGFVNRF112023 KTTTLILQKR KGFVRTALQH GAHLVPTFTF GETEVYDQVL FHKDSRMYKF 86606GKNAVTLRNR KGFVKLALRH GADLVPIYSF GENEVYKQVI FEEGSWGRWV 58765s GSFTLLLRNRKGFVRLALTH GYQASGKSTL G......SVG NWQG...FYF 58765 GSFTLLLRNR KGFVRLALTHGAPLVPIFSF GENDLFDQIP NSSGSWLRYI 112037 QEALRP.... LLSVALQLFL GRR......GLPLPFRAPIR TVVGSAIPVQ 60489 QLTFKK.... LMGFSPCIFW GRGLFSATSW GLLPFAVPITTVVGRPIPVP DC2 QNKLQK.... IMGFALPLFH ARG.VFQYNF GLMTYRKAIH TVVGRPIPVR112041 QKTFQDTFKK ILGLNFCTFH GRG.FTRGSW GFLPFNRPIT TVVGEPLPIP 112024QKWFQS.... MVHIYPCAFY GRG.FTKNSW GLLPYSRPVT TIVGEPLPMP 112023 QSCFRR....IFGFYCCVFY GQS.FCQGST GLLPYSRPIV TVVGEPLPLP 86606 QKKFQK.... YIGFAPCIFHGRGLFSSDTW GLVPYSKPIT TVVGEPITIP 58765s GGKMAE.... TNADSI.... ...........LVEIFSPFT IKIIFWCLMP 58765 QNRLQK.... IMGISLPLFH GRG.VFQYSF GLIPYRRPITTVVGKPIEVQ 112037 QSPPPSPAQV DTLQARYVGR LTQLFEEHQA RYGVPADRHL VLTEARPTAWPRLSAG 60489 QRLHPTEEEV NHYHALYMTA LEQLFEEHKE SCGVPASTCL TFI............. DC2 QTLNPTQEQI EELHQTYMEE LRKLFEEHKG KYGIPEHETL VLK....... ......112041 RIKRPNQKTV DKYHALYISA LRKLFDQHKV EYGLPETQEL TIT....... ......112024 KIENPSQEIV AKYHTLYIDA LRKLFDQHKT KFGISETQEL EII....... ......112023 QIEKPSQEMV DKYHALYMDA LDKLFDQHKT HYGCSETQKL FFL....... ......86606 KLEHPTQQDI DLYHTMYMEA LVKLFDKHKT KFGLPETEVL EVN....... ......58765s KYLEKEP... ....QRRLSD LRN....... .......... .......... ......58765 KTLHPSEEEV NQLHQRYIKE LCNLFEAHKL KFNIPADQHL EFC....... ......

The percent identity of the DGAT2 family members ranges from 33%identity (e.g., 112024 and 58765s share 33% identity) to 75% identity(e.g., 58765 short and long forms share 75% identity). The majority ofthe full length sequences share between 44% to 51% identity (e.g.,112041 and 60489; 112023 and 60489; as well as 86606 and 58765 share 44%identity; 112041 and 112024, 112041 and 112023, 112024 and 112023,112023 and 86606 share 51% identity) over their entire lengths. The mostclosely related full length human family members are 58765 and DC2,which share 52% identity over their entire lengths.

In one embodiment, a DGAT2 family member protein includes at least onetransmembrane domain. As used herein, the term “transmembrane domain”includes an amino acid sequence of about 15 amino acid residues inlength that spans a phospholipid membrane. More preferably, atransmembrane domain includes about at least 16, 17, 18, 20, 21, 22, 23,or 24 amino acid residues and spans a phospholipid membrane.Transmembrane domains are rich in hydrophobic residues, and typicallyhave an α-helical structure. In a preferred embodiment, at least 50%,60%, 70%, 80%, 90%, 95% or more of the amino acids of a transmembranedomain are hydrophobic, e.g., leucines, isoleucines, tyrosines, ortryptophans. Transmembrane domains are described in, for example,Zagotta W. N. et al., (1996) Annual Rev. Neuronsci. 19: 235-63, thecontents of which are incorporated herein by reference.

In a preferred embodiment, a DGAT2 family member polypeptide or proteinhas at least one transmembrane domain or a region which includes atleast 16, 17, 18, 20, 21, 22, 23, or 24 amino acid residues and has atleast about 60%, 70% 80% 90% 95%, 99%, or 100% homology with a“transmembrane domain,” e.g., at least one transmembrane domain of humanDGAT2 family member.

In another embodiment, a DGAT2 family member protein includes at leastone “non-transmembrane domain.” As used herein, “non-transmembranedomains” are domains that reside outside of the membrane. When referringto plasma membranes, non-transmembrane domains include extracellulardomains (i.e., outside of the cell) and intracellular domains (i.e.,within the cell). When referring to membrane-bound proteins found inintracellular organelles (e.g., mitochondria, endoplasmic reticulum,peroxisomes and microsomes), non-transmembrane domains include thosedomains of the protein that reside in the cytosol (i.e., the cytoplasm),the lumen of the organelle, or the matrix or the intermembrane space(the latter two relate specifically to mitochondria organelles). TheC-terminal amino acid residue of a non-transmembrane domain is adjacentto an N-terminal amino acid residue of a transmembrane domain in anaturally-occurring DGAT2 family member, or DGAT2 family member-likeprotein.

In a preferred embodiment, a DGAT2 family member polypeptide or proteinhas a “non-transmembrane domain” or a region which includes at leastabout 1-100, preferably about 2-80, more preferably about 5-70, and evenmore preferably about 8-65 amino acid residues, and has at least about60%, 70% 80% 90% 95%, 99% or 100% homology with a “non-transmembranedomain”, e.g., a non-transmembrane domain of human DGAT2 family member.Preferably, a non-transmembrane domain is capable of catalytic activity.

As the DGAT2 family member polypeptides of the invention may modulateDGAT2 family member-mediated activities (e.g., triglyceride synthesis),they may be useful for developing novel diagnostic and therapeuticagents for DGAT2 family member-mediated or related disorders (e.g.,obesity, triglyceride deficiency), as described below.

As used herein, a “DGAT2 family member activity”, “biological activityof DGAT2 family member” or “functional activity of DGAT2 family member”,refers to an activity exerted by a DGAT2 family member protein,polypeptide or nucleic acid molecule on e.g., a DGAT2 familymember-responsive cell or on a DGAT2 family member substrate, e.g., adiacylglycerol substrate, as determined in vivo or in vitro. In oneembodiment, a DGAT2 family member activity is a direct activity, such asan association with a DGAT2 family member target molecule. A “targetmolecule” or “binding partner” is a molecule with which a DGAT2 familymember protein binds or interacts in nature (e.g., diacylglycerol,acyl-coA). A DGAT2 family member activity can also be an indirectactivity, e.g., accumulation of fat stores as result of the DGAT2 familymember activity.

The DGAT2 family member molecules of the present invention are predictedto have similar biological activities as DGAT2 family members. Forexample, the DGAT2 family member proteins of the present invention canhave one or more of the following activities: (1) regulating, sensingand/or producing triglycerides in a cell, (for example, a fat cell(e.g., an adipocyte), a liver cell (e.g., a hepatocyte), a smallintestine cell); (2) interacting with (e.g., binding to) a diglyceridemolecule; (3) mobilizing an intracellular molecule that participates ina triglyceride biosynthesis (e.g., diacylglycerol or acyl-coA); (4)regulating diglyceride utilization; (5) altering the structure orcomponents of a cell (e.g., and adipocyte); and (6) modulating cellproliferation; migration, cell differentiation; and cell survival. Thus,the DGAT2 family member molecules can act as novel diagnostic targetsand therapeutic agents for controlling DGAT2 family member-relateddisorders (e.g., obesity and related disorders). Other activities, asdescribed below, include the ability to modulate function, survival,morphology, proliferation and/or differentiation of cells of tissues inwhich DGAT2 family member molecules are expressed (e.g., adipocytes).

The response mediated by a DGAT2 family member receptor protein dependson the type of cell. For example, in some cells, binding of a ligand tothe receptor protein may stimulate an activity such as release ofcompounds, gating of a channel, cellular adhesion, migration,differentiation, etc., through phosphatidylinositol or cyclic AMPmetabolism and turnover while in other cells, the binding of the ligandwill produce a different result. Regardless of the cellularactivity/response modulated by the protein, it is universal that theprotein is a DGAT2 family member and interacts with substrate (e.g.,acyl-coA, acylglycerol) to produce triacylglycerol in a cell. As usedherein, a “triacylglycerol biosynthesis” or “triglyceride biosynthesis”refers to the modulation (e.g., stimulation or inhibition) of a cellularfunction/activity upon the binding of a substrate to the DGAT2 familymember (DGAT2 family member protein). Examples of such functions includemobilization of lipid in adipocytes, production of fat stores.

Based on the above-described sequence similarities, the DGAT2 familymember molecules of the present invention are predicted to have similarbiological activities as diacylglycerol transferase family members.Thus, the DGAT2 family member molecules can act as novel diagnostictargets and therapeutic agents for controlling one or more of disordersassociated with adipocyte differentiation and metabolism and metabolicdisorders, cardiovascular disorders, liver disorders, cellularproliferative and/or differentiative disorders, or viral diseases.

The present invention is based, at least in part, on the discovery thatthe DGAT2 family member nucleic acid and polypeptide molecules areexpressed at high levels in adipose, liver, small intestine tissue, areregulated during conditions which affect differentiation and metabolismof adipocytes, and are downregulated in genetic animal models of obesity(see Examples and Tables described herein). Without intending to belimited by mechanism, it is believed that DGAT2 family member moleculescan modulate the metabolism by (directly or indirectly) affecting therate of lipogenesis and/or lipolysis, and production and maintenance offat storage in mammals.

As used herein, the term “metabolic disorder” includes a disorder,disease or condition which is caused or characterized by an abnormalmetabolism (i.e., the chemical changes in living cells by which energyis provided for vital processes and activities) in a subject. Metabolicdisorders include diseases, disorders, or conditions associated withaberrant thermogenesis or aberrant adipose cell (e.g., brown or whiteadipose cell) content or function. Metabolic disorders can becharacterized by a misregulation (e.g., downregulation or upregulation)of DGAT2 family member activity. Metabolic disorders can detrimentallyaffect cellular functions such as cellular proliferation, growth,differentiation, or migration, cellular regulation of homeostasis,inter- or intra-cellular communication; tissue function, such as liverfunction, muscle function, or adipocyte function; systemic responses inan organism, such as hormonal responses (e.g., insulin response).Examples of metabolic disorders include obesity, diabetes (e.g.,diabetes insipidus, diabetes mellitus (type I), diabetes mellitus (typeII)), endocrine abnormalities, triglyceride storage disease,Bardet-Biedl syndrome, Lawrence-Moon syndrome, and Prader-Labhart-Willisyndrome. Obesity is defined as a body mass index (BMI) of 30 kg/²m ormore (National Institute of Health, Clinical Guidelines on theIdentification, Evaluation, and Treatment of Overweight and Obesity inAdults (1998)). However, the present invention is also intended toinclude a disease, disorder, or condition that is characterized by abody mass index (BMI) of 25 kg/²m or more, 26 kg/²m or more, 27 kg/²m ormore, 28 kg/2m or more, 29 kg/²m or more, 29.5 kg/²m or more, or 29.9kg/²m or more, all of which are typically referred to as overweight(National Institute of Health, Clinical Guidelines on theIdentification, Evaluation, and Treatment of Overweight and Obesity inAdults (1998)). Additional metabolic disorders include lipid disorders(e.g., familial hypercholesteroliemia, polygenic hypercholesteroliemia,familial hypertriglyceridemia, familial lipoprotein lipase deficiency,combined hyperlipidemia, dysbetalipoproteinemia, sitosterolemia, Tangierdisease, hypobetalipoproteinemia, lecithin:cholesterol acyltransferase(LCAT) deficiency, and cerebrotendinous xanthomatosis) and toxic andacquired metabolic diseases.

As used herein, disorders involving the heart, or “cardiovasculardisease” or a “cardiovascular disorder” includes a disease or disorderwhich affects the cardiovascular system, e.g., the heart, the bloodvessels, and/or the blood. A cardiovascular disorder can be caused by animbalance in arterial pressure, a malfunction of the heart, or anocclusion of a blood vessel, e.g., by a thrombus. A cardiovasculardisorder includes, but is not limited to disorders such asarteriosclerosis, atherosclerosis, cardiac hypertrophy, ischemiareperfusion injury, restenosis, arterial inflammation, vascular wallremodeling, ventricular remodeling, rapid ventricular pacing, coronarymicroembolism, tachycardia, bradycardia, pressure overload, aorticbending, coronary artery ligation, vascular heart disease, valvulardisease, including but not limited to, valvular degeneration caused bycalcification, rheumatic heart disease, endocarditis, or complicationsof artificial valves; atrial fibrillation, long-QT syndrome, congestiveheart failure, sinus node dysfunction, angina, heart failure,hypertension, atrial fibrillation, atrial flutter, pericardial disease,including but not limited to, pericardial effusion and pericarditis;cardiomyopathies, e.g., dilated cardiomyopathy or idiopathiccardiomyopathy, myocardial infarction, coronary artery disease, coronaryartery spasm, ischemic disease, arrhythmia, sudden cardiac death, andcardiovascular developmental disorders (e.g., arteriovenousmalformations, arteriovenous fistulae, raynaud's syndrome, neurogenicthoracic outlet syndrome, causalgia/reflex sympathetic dystrophy,hemangioma, aneurysm, cavernous angioma, aortic valve stenosis, atrialseptal defects, atrioventricular canal, coarctation of the aorta,ebsteins anomaly, hypoplastic left heart syndrome, interruption of theaortic arch, mitral valve prolapse, ductus arteriosus, patent foramenovale, partial anomalous pulmonary venous return, pulmonary atresia withventricular septal defect, pulmonary atresia without ventricular septaldefect, persistance of the fetal circulation, pulmonary valve stenosis,single ventricle, total anomalous pulmonary venous return, transpositionof the great vessels, tricuspid atresia, truncus arteriosus, ventricularseptal defects). A cardiovascular disease or disorder also can includean endothelial cell disorder.

As used herein, “liver disorders” which can be treated or diagnosed bymethods described herein include, but are not limited to, disordersassociated with an accumulation in the liver of fibrous tissue, such asthat resulting from an imbalance between production and degradation ofthe extracellular matrix accompanied by the collapse and condensation ofpreexisting fibers. The methods described herein can be used to diagnoseor treat hepatocellular necrosis or injury induced by a wide variety ofagents including processes which disturb homeostasis, such as aninflammatory process, tissue damage resulting from toxic injury oraltered hepatic blood flow, and infections (e.g., bacterial, viral andparasitic). For example, the methods can be used for the early detectionof hepatic injury, such as portal hypertension or hepatic fibrosis. Inaddition, the methods can be employed to detect liver fibrosisattributed to inborn errors of metabolism, for example, fibrosisresulting from a storage disorder such as Gaucher's disease (lipidabnormalities) or a glycogen storage disease, A1-antitrypsin deficiency;a disorder mediating the accumulation (e.g., storage) of an exogenoussubstance, for example, hemochromatosis (iron-overload syndrome) andcopper storage diseases (Wilson's disease), disorders resulting in theaccumulation of a toxic metabolite (e.g., tyrosinemia, fructosemia andgalactosemia) and peroxisomal disorders (e.g., Zellweger syndrome).Additionally, the methods described herein can be used for the earlydetection and treatment of liver injury associated with theadministration of various chemicals or drugs, such as for example,methotrexate, isonizaid, oxyphenisatin, methyldopa, chlorpromazine,tolbutamide or alcohol, or which represents a hepatic manifestation of avascular disorder such as obstruction of either the intrahepatic orextrahepatic bile flow or an alteration in hepatic circulationresulting, for example, from chronic heart failure, veno-occlusivedisease, portal vein thrombosis or Budd-Chiari syndrome.

Additionally, DGAT2 family member molecules can play an important rolein the etiology of certain viral diseases, including but not limited toHepatitis B, Hepatitis C and Herpes Simplex Virus (HSV). Modulators ofDGAT2 family member activity could be used to control viral diseases.The modulators can be used in the treatment and/or diagnosis of viralinfected tissue or virus-associated tissue fibrosis, especially liverand liver fibrosis. Also, DGAT2 family member modulators can be used inthe treatment and/or diagnosis of virus-associated carcinoma, especiallyhepatocellular cancer.

As used interchangeably herein, “DGAT2 family member activity,”“biological activity of DGAT2 family member” or “functional activity ofDGAT2 family member,” includes an activity exerted by a DGAT2 familymember protein, polypeptide or nucleic acid molecule on a DGAT2 familymember responsive cell or tissue, e.g., adipocytes, or on a DGAT2 familymember protein substrate, e.g., diacylglycerol, as determined in vivo,or in vitro, according to standard techniques. DGAT2 familymember-mediated function can include modulation of metabolism. Examplesof such target molecules include proteins in the same biosynthetic pathas the DGAT2 family member protein, e.g., proteins which may functionupstream (including both stimulators and inhibitors of activity) ordownstream of the DGAT2 family member protein in a pathway involvingregulation of metabolism. The biological activities of DGAT2 familymember proteins can have one or more of the following activities: 1)modulation of fat homeostasis; 2) modulation of lipogenesis (e.g., fatdeposition necessary for heat insulation, mechanical cushion, and/orstorage); 3) modulation of lipolysis (e.g., fat mobilization necessaryas an energy source and/or for thermogenesis); and 4) modulation ofadipocyte growth (e.g., hyperplastic and/or hypertrophic growth).

As used herein, “metabolic activity” includes an activity exerted by anadipose cell, or an activity that takes place in an adipose cell. Forexample, such activities include cellular processes that contribute tothe physiological role of adipose cells, such as lipogenesis andlipolysis and include, but are not limited to, cell proliferation,differentiation, growth, migration, programmed cell death, uncoupledmitochondrial respiration, and thermogenesis.

The DGAT2 family member proteins, fragments thereof, and derivatives andother variants of the sequences in SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ IDNO:62 are collectively referred to as “polypeptides or proteins of theinvention” or “DGAT2 family member polypeptides or proteins”. Nucleicacid molecules encoding such polypeptides or proteins are collectivelyreferred to as “nucleic acids of the invention” or “DGAT2 family membernucleic acids.” DGAT2 family member molecules refer to DGAT2 familymember nucleic acids, polypeptides, and antibodies.

As used herein, the term “nucleic acid molecule” includes DNA molecules(e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) andanalogs of the DNA or RNA generated, e.g., by the use of nucleotideanalogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

The term “isolated or purified nucleic acid molecule” includes nucleicacid molecules which are separated from other nucleic acid moleculeswhich are present in the natural source of the nucleic acid. Forexample, with regards to genomic DNA, the term “isolated” includesnucleic acid molecules which are separated from the chromosome withwhich the genomic DNA is naturally associated. Preferably, an “isolated”nucleic acid is free of sequences which naturally flank the nucleic acid(i.e., sequences located at the 5′ and/or 3′ ends of the nucleic acid)in the genomic DNA of the organism from which the nucleic acid isderived. For example, in various embodiments, the isolated nucleic acidmolecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5kb or 0.1 kb of 5′ and/or 3′ nucleotide sequences which naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. Moreover, an “isolated” nucleic acid molecule,such as a cDNA molecule, can be substantially free of other cellularmaterial, or culture medium when produced by recombinant techniques, orsubstantially free of chemical precursors or other chemicals whenchemically synthesized.

As used herein, the term “hybridizes under stringent conditions”describes conditions for hybridization and washing. Stringent conditionsare known to those skilled in the art and can be found in CurrentProtocols in Molecular Biology, John Wiley & Sons, N.Y. (1989),6.3.1-6.3.6. Aqueous and nonaqueous methods are described in thatreference and either can be used. A preferred, example of stringenthybridization conditions are hybridization in 6× sodium chloride/sodiumcitrate (SSC) at about 45° C., followed by one or more washes in0.2×SSC, 0.1% SDS at 50° C. Another example of stringent hybridizationconditions are hybridization in 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by one or more washes in 0.2×SSC, 0.1% SDS at55° C. A further example of stringent hybridization conditions arehybridization in 6× sodium chloride/sodium citrate (SSC) at about 45°C., followed by one or more washes in 0.2×SSC, 0.1% SDS at 60° C.Preferably, stringent hybridization conditions are hybridization in 6×sodium chloride/sodium citrate (SSC) at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 65° C. Particularly preferredstringency conditions (and the conditions that should be used if thepractitioner is uncertain about what conditions should be applied todetermine if a molecule is within a hybridization limitation of theinvention) are 0.5M Sodium Phosphate, 7% SDS at 65° C., followed by oneor more washes at 0.2×SSC, 1% SDS at 65° C. Preferably, an isolatednucleic acid molecule of the invention that hybridizes under stringentconditions to the sequence of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:61,corresponds to a naturally-occurring nucleic acid molecule.

As used herein, a “naturally-occurring” nucleic acid molecule refers toan RNA or DNA molecule having a nucleotide sequence that occurs innature (e.g., encodes a natural protein).

As used herein, the terms “gene” and “recombinant gene” refer to nucleicacid molecules which include an open reading frame encoding a DGAT2family member protein, preferably a mammalian DGAT2 family memberprotein, and can further include non-coding regulatory sequences, andintrons.

An “isolated” or “purified” polypeptide or protein is substantially freeof cellular material or other contaminating proteins from the cell ortissue source from which the protein is derived, or substantially freefrom chemical precursors or other chemicals when chemically synthesized.In one embodiment, the language “substantially free” means preparationof DGAT2 family member protein having less than about 30%, 20%, 10% andmore preferably 5% (by dry weight), of non-DGAT2 family member protein(also referred to herein as a “contaminating protein”), or of chemicalprecursors or non-DGAT2 family member chemicals. When the DGAT2 familymember protein or biologically active portion thereof is recombinantlyproduced, it is also preferably substantially free of culture medium,i.e., culture medium represents less than about 20%, more preferablyless than about 10%, and most preferably less than about 5% of thevolume of the protein preparation. The invention includes isolated orpurified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams indry weight.

A “non-essential” amino acid residue is a residue that can be alteredfrom the wild-type sequence of DGAT2 family member (e.g., the sequenceof SEQ ID NO:7, SEQ ID NO:19, or SEQ ID NO:61 without abolishing or morepreferably, without substantially altering a biological activity,whereas an “essential” amino acid residue results in such a change. Forexample, amino acid residues that are conserved among the polypeptidesof the present invention, are predicted to be particularly unamenable toalteration.

A “conservative amino acid substitution” is one in which the amino acidresidue is replaced with an amino acid residue having a similar sidechain. Families of amino acid residues having similar side chains havebeen defined in the art. These families include amino acids with basicside chains (e.g., lysine, arginine, histidine), acidic side chains(e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g.,glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine),nonpolar side chains (e.g., alanine, valine, leucine, isoleucine,proline, phenylalanine, methionine, tryptophan), beta-branched sidechains (e.g., threonine, valine, isoleucine) and aromatic side chains(e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, apredicted nonessential amino acid residue in a DGAT2 family memberprotein is preferably replaced with another amino acid residue from thesame side chain family. Alternatively, in another embodiment, mutationscan be introduced randomly along all or part of a DGAT2 family membercoding sequence, such as by saturation mutagenesis, and the resultantmutants can be screened for DGAT2 family member biological activity toidentify mutants that retain activity. Following mutagenesis of a DGAT2family member nucleotide sequence of the invention, the encoded proteincan be expressed recombinantly and the activity of the protein can bedetermined.

As used herein, a “biologically active portion” of a DGAT2 family memberprotein includes a fragment of a DGAT2 family member protein whichparticipates in an interaction between a DGAT2 family member moleculeand a non-DGAT2 family member molecule. Biologically active portions ofa DGAT2 family member protein include peptides comprising amino acidsequences sufficiently homologous to or derived from the amino acidsequence of the DGAT2 family member protein, e.g., the amino acidsequence shown in SEQ ID NO:8, SEQ ID NO:20, or SEQ ID NO:62 whichinclude less amino acids than the full length DGAT2 family memberproteins, and exhibit at least one activity of a DGAT2 family memberprotein. Typically, biologically active portions comprise a domain ormotif with at least one activity of the DGAT2 family member protein,e.g., diacylglycerol acyltransferase activity. A biologically activeportion of a DGAT2 family member protein can be a polypeptide which is,for example, 10, 25, 50, 100, 200 or more amino acids in length.Biologically active portions of a DGAT2 family member protein can beused as targets for developing agents which modulate a DGAT2 familymember mediated activity, e.g., diacylglycerol acyltransferase activity.

Calculations of homology or sequence identity between sequences (theterms are used interchangeably herein) are performed as follows.

To determine the percent identity of two amino acid sequences, or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence aligned forcomparison purposes is at least 30%, preferably at least 40%, morepreferably at least 50%, even more preferably at least 60%, and evenmore preferably at least 70%, 80%, 90%, 100% of the length of thereference sequence amino acid residues are aligned. The amino acidresidues or nucleotides at corresponding amino acid positions ornucleotide positions are then compared. When a position in the firstsequence is occupied by the same amino acid residue or nucleotide as thecorresponding position in the second sequence, then the molecules areidentical at that position (as used herein amino acid or nucleic acid“identity” is equivalent to amino acid or nucleic acid “homology”). Thepercent identity between the two sequences is a function of the numberof identical positions shared by the sequences, taking into account thenumber of gaps, and the length of each gap, which need to be introducedfor optimal alignment of the two sequences.

The comparison of sequences and determination of percent identitybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent identity between twoamino acid sequences is determined using the Needleman and Wunsch (J.Mol. Biol. (48):444-453 (1970)) algorithm which has been incorporatedinto the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent identity between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularlypreferred set of parameters (and the one that should be used if thepractitioner is uncertain about what parameters should be applied todetermine if a molecule is within a sequence identity or homologylimitation of the invention) is using a Blossum 62 scoring matrix with agap open penalty of 12, a gap extend penalty of 4, and a frameshift gappenalty of 5.

The percent identity between two amino acid or nucleotide sequences canbe determined using the algorithm of E. Meyers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences described herein can be used as a“query sequence” to perform a search against public databases to, forexample, identify other family members or related sequences. Suchsearches can be performed using the NBLAST and XBLAST programs (version2.0) of Altschul, et al., (1990) J. Mol. Biol. 215:403-10. BLASTnucleotide searches can be performed with the NBLAST program, score=100,wordlength=12 to obtain nucleotide sequences homologous to DGAT2 familymember nucleic acid molecules of the invention. BLAST protein searchescan be performed with the XBLAST program, score=50, wordlength=3 toobtain amino acid sequences homologous to DGAT2 family member proteinmolecules of the invention. To obtain gapped alignments for comparisonpurposes, Gapped BLAST can be utilized as described in Altschul et al.,(1997) Nucleic Acids Res. 25(17):3389-3402. When utilizing BLAST andGapped BLAST programs, the default parameters of the respective programs(e.g., XBLAST and NBLAST) can be used.

“Misexpression or aberrant expression”, as used herein, refers to anon-wild type pattern of gene expression, at the RNA or protein level.It includes: expression at non-wild type levels, i.e., over or underexpression; a pattern of expression that differs from wild type in termsof the time or stage at which the gene is expressed, e.g., increased ordecreased expression (as compared with wild type) at a predetermineddevelopmental period or stage; a pattern of expression that differs fromwild type in terms of decreased expression (as compared with wild type)in a predetermined cell type or tissue type; a pattern of expressionthat differs from wild type in terms of the splicing size, amino acidsequence, post-transitional modification, or biological activity of theexpressed polypeptide; a pattern of expression that differs from wildtype in terms of the effect of an environmental stimulus orextracellular stimulus on expression of the gene, e.g., a pattern ofincreased or decreased expression (as compared with wild type) in thepresence of an increase or decrease in the strength of the stimulus.

“Subject”, as used herein, can refer to an animal, e.g., a human, or anon-human mammal, e.g., a mouse, a rat, a primate, a horse, a cow, agoat, or other animal.

A “purified preparation of cells”, as used herein, refers to, in thecase of plant or animal cells, an in vitro preparation of cells and notan entire intact plant or animal. In the case of cultured cells ormicrobial cells, it consists of a preparation of at least 10% and morepreferably 50% of the subject cells.

Various aspects of the invention are described in further detail below.

Isolated Nucleic Acid Molecules

In one aspect, the invention provides, an isolated or purified, nucleicacid molecule that encodes a DGAT2 family member polypeptide describedherein, e.g., a full length DGAT2 family member protein or a fragmentthereof, e.g., a biologically active portion of DGAT2 family memberprotein. Also included is a nucleic acid fragment suitable for use as ahybridization probe, which can be used, e.g., to a identify nucleic acidmolecule encoding a polypeptide of the invention, DGAT2 family membermRNA, and fragments suitable for use as primers, e.g., PCR primers forthe amplification or mutation of nucleic acid molecules.

In one embodiment, an isolated nucleic acid molecule of the inventionincludes the nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, and SEQID NO:61 or a portion of any of these nucleotide sequences. In oneembodiment, the nucleic acid molecule includes sequences encoding theDGAT2 family member protein (e.g., “the coding region”, from nucleotides154-1194 of SEQ ID NO:7, not including the terminal codon), as well as5′ untranslated sequences (nucleotides 1-153 of SEQ ID NO:7).Alternatively, the nucleic acid molecule can include only the codingregion (e.g., nucleotides 154-1194 of SEQ ID NO:7) and, e.g., noflanking sequences which normally accompany the subject sequence. Inanother embodiment, the nucleic acid molecule encodes a sequencecorresponding to the mature protein of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO12, SEQ ID NO:14, SEQ IDNO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ IDNO:62.

In another embodiment, an isolated nucleic acid molecule of theinvention includes a nucleic acid molecule which is a complement of thenucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQ IDNO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:61 or aportion of any of these nucleotide sequences. In other embodiments, thenucleic acid molecule of the invention is sufficiently complementary tothe nucleotide sequence shown in SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, or SEQ ID NO:61,such that it can hybridize to the nucleotide sequence shown in SEQ IDNO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:1,SEQ ID NO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21,SEQ ID NO:23, or SEQ ID NO:61, thereby forming a stable duplex.

In one embodiment, an isolated nucleic acid molecule of the presentinvention includes a nucleotide sequence which is at least about 60%,65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, or more homologous to the nucleotide sequence shown in SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ IDNO:23 or SEQ ID NO:61. In the case of an isolated nucleic acid moleculewhich is longer than or equivalent in length to the reference sequence,e.g., SEQ ID NO:7, SEQ ID NO:19, the comparison is made with the fulllength of the reference sequence. Where the isolated nucleic acidmolecule is shorter than the reference sequence (e.g., shorter than SEQID NO:7, SEQ ID NO:19, SEQ ID NO:61), the comparison is made to asegment of the reference sequence of the same length (excluding any looprequired by the homology calculation).

DGAT2 family Member Nucleic Acid Fragments

A nucleic acid molecule of the invention can include only a portion ofthe DGAT2 family member nucleic acid sequences of the invention (e.g.,SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:61). For example, such a nucleicacid molecule can include a fragment which can be used as a probe orprimer or a fragment encoding a portion of a DGAT2 family memberprotein, e.g., an immunogenic or biologically active portion of a DGAT2family member protein. A fragment can comprise: nucleotides which encodea diacylglycerol acyltransferase domain of human DGAT2 family member.The nucleotide sequences determined from the cloning of the DGAT2 familymember genes allows for the generation of probes and primers designedfor use in identifying and/or cloning of additional DGAT2 family memberfamily members, or fragments thereof, as well as additional DGAT2 familymember homologues, or fragments thereof, from other species.

In another embodiment, a nucleic acid includes a nucleotide sequencethat includes part, or all, of the coding region and extends into either(or both) the 5′ or 3′ noncoding region. Other embodiments include afragment which includes a nucleotide sequence encoding an amino acidfragment described herein. Nucleic acid fragments can encode a specificdomain or site described herein or fragments thereof, particularlyfragments thereof which are at least 150 amino acids in length.Fragments also include nucleic acid sequences corresponding to specificamino acid sequences described above or fragments thereof. Nucleic acidfragments should not to be construed as encompassing those fragmentsthat may have been disclosed prior to the invention.

A nucleic acid fragment can include a sequence corresponding to adomain, region, or functional site described herein. A nucleic acidfragment can also include one or more domain, region, or functional sitedescribed herein. Thus, for example, the nucleic acid fragment caninclude a diacylglycerol acyltransferase domain. In a preferredembodiment the fragment is at least, 50, 100, 200, 300, 400, 500, 600,700, or 900 base pairs in length.

DGAT2 family member probes and primers are provided. Typically aprobe/primer is an isolated or purified oligonucleotide. Theoligonucleotide typically includes a region of nucleotide sequence thathybridizes under stringent conditions to at least about 7, 12 or 15,preferably about 20 or 25, more preferably about 30, 35, 40, 45, 50, 55,60, 65, or 75 consecutive nucleotides of a sense or antisense sequenceof the DGAT2 family member nucleic acid sequences of the invention(e.g., SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:61), or of a naturallyoccurring allelic variant or mutant of DGAT2 family member nucleic acidsequences of the invention (e.g., SEQ ID NO:7, SEQ ID NO:19, SEQ IDNO:61).

In a preferred embodiment the nucleic acid is a probe which is at least5 or 10, and less than 200, more preferably less than 100, or less than50, base pairs in length. It should be identical, or differ by 1, orless than in 5 or 10 bases, from a sequence disclosed herein. Ifalignment is needed for this comparison the sequences should be alignedfor maximum homology. “Looped” out sequences from deletions orinsertions, or mismatches, are considered differences.

A probe or primer can be derived from the sense or anti-sense strand ofa nucleic acid which encodes a diacylglycerol acyltransferase domain.

In another embodiment a set of primers is provided, e.g., primerssuitable for use in a PCR, which can be used to amplify a selectedregion of a DGAT2 family member sequence, e.g., a region describedherein. The primers should be at least 5, 10, or 50 base pairs in lengthand less than 100, or less than 200, base pairs in length. The primersshould be identical, or differ by one base from a sequence disclosedherein or from a naturally occurring variant. E.g., primers suitable foramplifying all or a portion of any of the following regions or domainsdescribed herein are provided (e.g., a diacylglycerol acyltransferasedomain).

A nucleic acid fragment can encode an epitope bearing region of apolypeptide described herein.

A nucleic acid fragment encoding a “biologically active portion of aDGAT2 family member polypeptide” can be prepared by isolating a portionof the nucleotide sequence of the DGAT2 family member sequences of theinvention (e.g., SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:61), which encodesa polypeptide having a DGAT2 family member biological activity (e.g.,the biological activities of the DGAT2 family member proteins asdescribed herein), expressing the encoded portion of the DGAT2 familymember protein (e.g., by recombinant expression in vitro) and assessingthe activity of the encoded portion of the DGAT2 family member protein.For example, a nucleic acid fragment encoding a biologically activeportion of DGAT2 family member includes a diacylglycerol acyltransferasedomain. A nucleic acid fragment encoding a biologically active portionof a DGAT2 family member polypeptide, may comprise a nucleotide sequencewhich is greater than 300-1200 or more nucleotides in length.

In preferred embodiments, nucleic acids include a nucleotide sequencewhich is about 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200,1300, 1400 nucleotides in length and hybridizes under stringenthybridization conditions to a nucleic acid molecule of DGAT2 familymember nucleic acid sequences of the invention (e.g., SEQ ID NO:7, SEQID NO:19, SEQ ID NO:61).

DGAT2 Family Member Nucleic Acid Variants

The invention further encompasses nucleic acid molecules that differfrom the DGAT2 family member nucleotide sequences of the invention(e.g., SEQ ID NO:7, SEQ ID NO:19, SEQ ID NO:61). Such differences can bedue to degeneracy of the genetic code (and result in a nucleic acidwhich encodes the same DGAT2 family member proteins as those encoded bythe nucleotide sequence disclosed herein. In another embodiment, anisolated nucleic acid molecule of the invention has a nucleotidesequence encoding a protein having an amino acid sequence which differs,by at least 1, but less than 5, 10, 20, 50, or 100 amino acid residuesof the DGAT2 family member protein sequences provided (e.g. SEQ ID NO:8,SEQ ID NO:20, SEQ ID NO:62). If alignment is needed for this comparisonthe sequences should be aligned for maximum homology. “Looped” outsequences from deletions or insertions, or mismatches, are considereddifferences.

Nucleic acids of the inventor can be chosen for having codons, which arepreferred, or non preferred, for a particular expression system. E.g.,the nucleic acid can be one in which at least one colon, at preferablyat least 10%, or 20% of the codons has been altered such that thesequence is optimized for expression in E. coli, yeast, human, insect,or CHO cells.

Nucleic acid variants can be naturally occurring, such as allelicvariants (same locus), homologs (different locus), and orthologs(different organism) or can be non-naturally occurring. Non-naturallyoccurring variants can be made by mutagenesis techniques, includingthose applied to polynucleotides, cells, or organisms. The variants cancontain nucleotide substitutions, deletions, inversions and insertions.Variation can occur in either or both the coding and non-coding regions.The variations can produce both conservative and non-conservative aminoacid substitutions (as compared in the encoded product).

In a preferred embodiment, the nucleic acid differs from that of thenucleic acid sequences of the invention (e.g., SEQ ID NO:7, SEQ IDNO:19, SEQ ID NO:61), e.g., as follows: by at least one but less than10, 20, 30, or 40 nucleotides; at least one but less than 1%, 5%, 10% or20% of the in the subject nucleic acid. If necessary for this analysisthe sequences should be aligned for maximum homology. “Looped” outsequences from deletions or insertions, or mismatches, are considereddifferences.

Orthologs, homologs, and allelic variants can be identified usingmethods known in the art. These variants comprise a nucleotide sequenceencoding a polypeptide that is 50%, at least about 55%, typically atleast about 70-75%, more typically at least about 80-85%, and mosttypically at least about 90-95% or more identical to the amino acidsequences of the invention (e.g., SEQ ID NO:8, SEQ ID NO:20, SEQ IDNO:62) or a fragment of those sequences. Such nucleic acid molecules canreadily be obtained as being able to hybridize under stringentconditions, to the nucleotide sequence shown in SEQ ID NO:7, SEQ IDNO:19, or SEQ ID NO:61 or a fragment of this sequence. Nucleic acidmolecules corresponding to orthologs, homologs, and allelic variants ofthe DGAT2 family member cDNAs of the invention can further be isolatedby mapping to the same chromosome or locus as the DGAT2 family membergene. Preferred variants include those that are correlated withdiacylglycerol acyltransferase activity.

Allelic variants of DGAT2 family member, e.g., human DGAT2 familymember, include both functional and non-functional proteins. Functionalallelic variants are naturally occurring amino acid sequence variants ofthe DGAT2 family member protein within a population that maintain theability to modulate the phosphorylation state of itself or anotherprotein or polypeptide. Functional allelic variants will typicallycontain only conservative substitution of one or more amino acids of theDGAT2 family member amino acid sequences of the invention (e.g., SEQ IDNO:8 or SEQ ID NO:20 or SEQ ID NO:62), or substitution, deletion orinsertion of non-critical residues in non-critical regions of theprotein. Non-functional allelic variants are naturally-occurring aminoacid sequence variants of the DGAT2 family member, e.g., human DGAT2family member, protein within a population that do not have the abilityto attach an acyl chain to a lipid precursor. Non-functional allelicvariants will typically contain a non-conservative substitution, adeletion, or insertion, or premature truncation of the amino acidsequences of the invention (e.g., SEQ ID NO:8 or SEQ ID NO:20 or SEQ IDNO:62), or a substitution, insertion, or deletion in critical residuesor critical regions of the protein.

Moreover, nucleic acid molecules encoding other DGAT2 family memberfamily members and, thus, which have a nucleotide sequence which differsfrom the DGAT2 family member sequences of the invention (e.g., SEQ IDNO:7, SEQ ID NO:19 or SEQ ID NO:61) are intended to be within the scopeof the invention.

Antisense Nucleic Acid Molecules, Ribozymes and Modified DGAT2 FamilyMember Nucleic Acid Molecules

In another aspect, the invention features, an isolated nucleic acidmolecule which is antisense to DGAT2 family member. An “antisense”nucleic acid can include a nucleotide sequence which is complementary toa “sense” nucleic acid encoding a protein, e.g., complementary to thecoding strand of a double-stranded cDNA molecule or complementary to anmRNA sequence. The antisense nucleic acid can be complementary to anentire DGAT2 family member coding strand, or to only a portion thereof(e.g., the coding region of human DGAT2 family member corresponding toDGAT2 family member sequences of the invention, e.g., SEQ ID NO:7 SEQ IDNO:19 or SEQ ID NO:61). In another embodiment, the antisense nucleicacid molecule is antisense to a “noncoding region” of the coding strandof a nucleotide sequence encoding DGAT2 family member (e.g., the 5′ and3′ untranslated regions).

An antisense nucleic acid can be designed such that it is complementaryto the entire coding region of DGAT2 family member mRNA, but morepreferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of DGAT2 family member mRNA. For example,the antisense oligonucleotide can be complementary to the regionsurrounding the translation start site of DGAT2 family member mRNA,e.g., between the −10 and +10 regions of the target gene nucleotidesequence of interest. An antisense oligonucleotide can be, for example,about 7, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, ormore nucleotides in length.

An antisense nucleic acid of the invention can be constructed usingchemical synthesis and enzymatic ligation reactions using proceduresknown in the art. For example, an antisense nucleic acid (e.g., anantisense oligonucleotide) can be chemically synthesized using naturallyoccurring nucleotides or variously modified nucleotides designed toincrease the biological stability of the molecules or to increase thephysical stability of the duplex formed between the antisense and sensenucleic acids, e.g., phosphorothioate derivatives and acridinesubstituted nucleotides can be used. The antisense nucleic acid also canbe produced biologically using an expression vector into which a nucleicacid has been subcloned in an antisense orientation (i.e., RNAtranscribed from the inserted nucleic acid will be of an antisenseorientation to a target nucleic acid of interest, described further inthe following subsection).

The antisense nucleic acid molecules of the invention are typicallyadministered to a subject (e.g., by direct injection at a tissue site),or generated in situ such that they hybridize with or bind to cellularmRNA and/or genomic DNA encoding a DGAT2 family member protein tothereby inhibit expression of the protein, e.g., by inhibitingtranscription and/or translation. Alternatively, antisense nucleic acidmolecules can be modified to target selected cells and then administeredsystemically. For systemic administration, antisense molecules can bemodified such that they specifically bind to receptors or antigensexpressed on a selected cell surface, e.g., by linking the antisensenucleic acid molecules to peptides or antibodies which bind to cellsurface receptors or antigens. The antisense nucleic acid molecules canalso be delivered to cells using the vectors described herein. Toachieve sufficient intracellular concentrations of the antisensemolecules, vector constructs in which the antisense nucleic acidmolecule is placed under the control of a strong pol II or pol IIIpromoter are preferred.

In yet another embodiment, the antisense nucleic acid molecule of theinvention is an α-anomeric nucleic acid molecule. An α-anomeric nucleicacid molecule forms specific double-stranded hybrids with complementaryRNA in which, contrary to the usual β-units, the strands run parallel toeach other (Gaultier et al., (1987) Nucleic Acids. Res. 15:6625-6641).The antisense nucleic acid molecule can also comprise a2′-o-methylribonucleotide (Inoue et al., (1987) Nucleic Acids Res.15:6131-6148) or a chimeric RNA-DNA analogue (Inoue et al., (1987) FEBSLett. 215:327-330).

In still another embodiment, an antisense nucleic acid of the inventionis a ribozyme. A ribozyme having specificity for a DGAT2 familymember-encoding nucleic acid can include one or more sequencescomplementary to the nucleotide sequence of a DGAT2 family member cDNAdisclosed herein (e.g., SEQ ID NO:7, SEQ ID NO:19 or SEQ ID NO:61), anda sequence having known catalytic sequence responsible for mRNA cleavage(see U.S. Pat. No. 5,093,246 or Haselhoff and Gerlach, (1988) Nature334:585-591). For example, a derivative of a Tetrahymena L-19 IVS RNAcan be constructed in which the nucleotide sequence of the active siteis complementary to the nucleotide sequence to be cleaved in a DGAT2family member-encoding mRNA. See, e.g., Cech et al. U.S. Pat. No.4,987,071; and Cech et al. U.S. Pat. No. 5,116,742. Alternatively, DGAT2family member mRNA can be used to select a catalytic RNA having aspecific ribonuclease activity from a pool of RNA molecules. See, e.g.,Bartel, D. and Szostak, J. W. (1993) Science 261:1411-1418.

DGAT2 family member gene expression can be inhibited by targetingnucleotide sequences complementary to the regulatory region of the DGAT2family member (e.g., the DGAT2 family member promoter and/or enhancers)to form triple helical structures that prevent transcription of theDGAT2 family member gene in target cells. See generally, Helene, C.,(1991) Anticancer Drug Des. 6(6):569-84; Helene, C. et al., (1992) Ann.N.Y. Acad. Sci. 660:27-36; and Maher, L. J., (1992) Bioassays14(12):807-15. The potential sequences that can be targeted for triplehelix formation can be increased by creating a so-called “switchback”nucleic acid molecule. Switchback molecules are synthesized in analternating 5′-3′,3′-5′ manner, such that they base pair with first onestrand of a duplex and then the other, eliminating the necessity for asizeable stretch of either purines or pyrimidines to be present on onestrand of a duplex.

The invention also provides detectably labeled oligonucleotide primerand probe molecules. Typically, such labels are chemiluminescent,fluorescent, radioactive, or colorimetric.

A DGAT2 family member nucleic acid molecule can be modified at the basemoiety, sugar moiety or phosphate backbone to improve, e.g., thestability, hybridization, or solubility of the molecule. For example,the deoxyribose phosphate backbone of the nucleic acid molecules can bemodified to generate peptide nucleic acids (see Hyrup B. et al., (1996)Bioorganic & Medicinal Chemistry 4 (1): 5-23). As used herein, the terms“peptide nucleic acid” or “PNA” refers to a nucleic acid mimic, e.g., aDNA mimic, in which the deoxyribose phosphate backbone is replaced by apseudopeptide backbone and only the four natural nucleobases areretained. The neutral backbone of a PNA can allow for specifichybridization to DNA and RNA under conditions of low ionic strength. Thesynthesis of PNA oligomers can be performed using standard solid phasepeptide synthesis protocols as described in Hyrup B. et al., (1996)supra; Perry-O'Keefe et al., Proc. Natl. Acad. Sci. 93: 14670-675.

PNAs of DGAT2 family member nucleic acid molecules can be used intherapeutic and diagnostic applications. For example, PNAs can be usedas antisense or antigene agents for sequence-specific modulation of geneexpression by, for example, inducing transcription or translation arrestor inhibiting replication. PNAs of DGAT2 family member nucleic acidmolecules can also be used in the analysis of single base pair mutationsin a gene, (e.g., by PNA-directed PCR clamping); as ‘artificialrestriction enzymes’ when used in combination with other enzymes, (e.g.,S1 nucleases (Hyrup B., (1996) supra)); or as probes or primers for DNAsequencing or hybridization (Hyrup B. et al., (1996) supra;Perry-O'Keefe supra).

In other embodiments, the oligonucleotide may include other appendedgroups such as peptides (e.g., for targeting host cell receptors invivo), or agents facilitating transport across the cell membrane (see,e.g., Letsinger et al., (1989) Proc. Natl. Acad. Sci. USA 86:6553-6556;Lemaitre et al., (1987) Proc. Natl. Acad. Sci. USA 84:648-652; PCTPublication No. WO88/09810) or the blood-brain barrier (see, e.g., PCTPublication No. WO89/10134). In addition, oligonucleotides can bemodified with hybridization-triggered cleavage agents (See, e.g., Krolet al., (1988) Bio-Techniques 6:958-976) or intercalating agents. (See,e.g., Zon, (1988) Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, (e.g., a peptide,hybridization triggered cross-linking agent, transport agent, orhybridization-triggered cleavage agent).

The invention also includes molecular beacon oligonucleotide primer andprobe molecules having at least one region which is complementary to aDGAT2 family member nucleic acid of the invention, two complementaryregions one having a fluorophore and one a quencher such that themolecular beacon is useful for quantitating the presence of the DGAT2family member nucleic acid of the invention in a sample. Molecularbeacon nucleic acids are described, for example, in Lizardi et al., U.S.Pat. No. 5,854,033; Nazarenko et al., U.S. Pat. No. 5,866,336, and Livaket al., U.S. Pat. No. 5,876,930.

Isolated DGAT2 Family Member Polypeptides

In another aspect, the invention features, an isolated DGAT2 familymember protein, or fragment, e.g., a biologically active portion, foruse as immunogens or antigens to raise or test (or more generally tobind) anti-DGAT2 family member antibodies. DGAT2 family member proteincan be isolated from cells or tissue sources using standard proteinpurification techniques. DGAT2 family member protein or fragmentsthereof can be produced by recombinant DNA techniques or synthesizedchemically.

Polypeptides of the invention include those which arise as a result ofthe existence of multiple genes, alternative transcription events,alternative RNA splicing events, and alternative translational andpostranslational events. The polypeptide can be expressed in systems,e.g., cultured cells, which result in substantially the samepostranslational modifications present when expressed the polypeptide isexpressed in a native cell, or in systems which result in the alterationor omission of postranslational modifications, e.g., gylcosylation orcleavage, present when expressed in a native cell.

In a preferred embodiment, a DGAT2 family member polypeptide has one ormore of the following characteristics:

it has the ability to regulate, sense and/or transmit an extracellularsignal into a cell; it has the ability to interact with (e.g., bind to)an extracellular signal or a cell surface receptor; it has the abilityto mobilize an intracellular molecule that participates in a signaltransduction pathway (e.g., adenylate cyclase or phosphatidylinositol4,5-bisphosphate (PIP₂), inositol 1,4,5-triphosphate (IP₃));it has the ability to regulate polarization of the plasma membrane;it has the ability to modulate cell proliferation, cell migration,differentiation and/or cell survival; it has the ability to modulatefunction, survival, morphology, proliferation and/or differentiation ofcells of tissues in which DGAT2 family member molecules are expressed;it has a molecular weight (e.g., deduced molecular weight), amino acidcomposition or other physical characteristic of a DGAT2 family memberprotein of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO12, SEQ ID NO:14, SEQ ID NO: 16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24, or SEQ ID NO:62;it has an overall sequence similarity (identity) of at least 60%,preferably at least 70%, more preferably at least 75, 80, 85, 86, 87,88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99% or more, with apolypeptide of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID N012, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:62; it has an N-terminaldomain which is preferably about 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%or higher, identical to a polypeptide of SEQ ID NO:2;it has at least one transmembrane domains which is preferably about 70%,80%, 90%, 95% or higher, identical to a polypeptide of SEQ ID NO:2;it has a C-terminal domain which is preferably about 70%, 80%, 90%, 95%,96%, 97%, 98%, 99% or higher, identical to a polypeptide of SEQ ID NO:2;orit has an diacylglycerol acyltransferase domain which preferably has anoverall sequence similarity of about 70%, 80%, 90% or 95% with aminoacid residues 32-278 of SEQ ID NO:2.

In a preferred embodiment the DGAT2 family member protein, or fragmentthereof, differs from the corresponding sequence in SEQ ID NO:2, SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO12, SEQ ID NO:14,SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 orSEQ ID NO:62. In one embodiment it differs by at least one but by lessthan 15, 10 or 5 amino acid residues. In another it differs from thecorresponding sequence in SEQ ID NO:2 by at least one residue but lessthan 20%, 15%, 10% or 5% of the residues in it differ from thecorresponding sequence in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:62. (Ifthis comparison requires alignment the sequences should be aligned formaximum homology. “Looped” out sequences from deletions or insertions,or mismatches, are considered differences.) The differences are,preferably, differences or changes at a non-essential residue or aconservative substitution. In a preferred embodiment the differences arenot in the diacylglycerol acyltransferase domain. In another preferredembodiment one or more differences are in non-active site residues, e.g.outside of the diacylglycerol acyltransferase domain.

Other embodiments include a protein that contain one or more changes inamino acid sequence, e.g., a change in an amino acid residue which isnot essential for activity. Such DGAT2 family member proteins differ inamino acid sequence from SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:62, yetretain biological activity.

In one embodiment, a biologically active portion of a DGAT2 familymember protein includes an diacylglycerol acyltransferase domain. Inanother embodiment, a biologically active portion of a DGAT2 familymember protein includes a MttB family UPF0032 domain. Moreover, otherbiologically active portions, in which other regions of the protein aredeleted, can be prepared by recombinant techniques and evaluated for oneor more of the functional activities of a native DGAT2 family memberprotein.

In a preferred embodiment, the DGAT2 family member protein has an aminoacid sequence shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO12, SEQ ID NO:14, SEQ ID NO:16, SEQ IDNO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:62. Inother embodiments, the DGAT2 family member protein is substantiallyidentical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID N012, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:62 and retains thefunctional activity of the protein of SEQ ID NO:2, as described indetail above. Accordingly, in another embodiment, the DGAT2 familymember protein is a protein which includes an amino acid sequence atleast about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or moreidentical to SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ IDNO:20, SEQ ID NO:22, SEQ ID NO:24 or SEQ ID NO:62.

DGAT2 Family Member Chimeric or Fusion Proteins

In another aspect, the invention provides DGAT2 family member chimericor fusion proteins. As used herein, a DGAT2 family member “chimericprotein” or “fusion protein” includes a DGAT2 family member polypeptidelinked to a non-DGAT2 family member polypeptide. A “non-DGAT2 familymember polypeptide” refers to a polypeptide having an amino acidsequence corresponding to a protein which is not substantiallyhomologous to the DGAT2 family member protein, e.g., a protein which isdifferent from the DGAT2 family member protein and which is derived fromthe same or a different organism. The DGAT2 family member polypeptide ofthe fusion protein can correspond to all or a portion e.g., a fragmentdescribed herein of a DGAT2 family member amino acid sequence of theinvention. In a preferred embodiment, a DGAT2 family member fusionprotein includes at least one (or two) biologically active portion of aDGAT2 family member protein. The non-DGAT2 family member polypeptide canbe fused to the N-terminus or C-terminus of the DGAT2 family memberpolypeptide.

The fusion protein can include a moiety which has a high affinity for aligand. For example, the fusion protein can be a GST-DGAT2 family memberfusion protein in which the DGAT2 family member sequences are fused tothe C-terminus of the GST sequences. Such fusion proteins can facilitatethe purification of a recombinant DGAT2 family member polypeptide.Alternatively, the fusion protein can be a DGAT2 family member proteincontaining a heterologous signal sequence at its N-terminus. In certainhost cells (e.g., mammalian host cells), expression and/or secretion ofDGAT2 family member can be increased through use of a heterologoussignal sequence.

Fusion proteins can include all or a part of a serum protein, e.g., anIgG constant region, or human serum albumin.

The DGAT2 family member fusion proteins of the invention can beincorporated into pharmaceutical compositions and administered to asubject in vivo. The DGAT2 family member fusion proteins can be used toaffect the bioavailability of a DGAT2 family member substrate. DGAT2family member fusion proteins may be useful therapeutically for thetreatment of disorders caused by, for example, (i) aberrant modificationor mutation of a gene encoding a DGAT2 family member protein; (ii)mis-regulation of the DGAT2 family member gene; and (iii) aberrantpost-translational modification of a DGAT2 family member protein.

Moreover, the DGAT2 family member-fusion proteins of the invention canbe used as immunogens to produce anti-DGAT2 family member antibodies ina subject, to purify DGAT2 family member ligands and in screening assaysto identify molecules which inhibit the interaction of DGAT2 familymember with a DGAT2 family member substrate.

Expression vectors are commercially available that already encode afusion moiety (e.g., a GST polypeptide). A DGAT2 family member-encodingnucleic acid can be cloned into such an expression vector such that thefusion moiety is linked in-frame to the DGAT2 family member protein.

Variants of DGAT2 Family Member Proteins

In another aspect, the invention also features a variant of a DGAT2family member polypeptide, e.g., which functions as an agonist(mimetics) or as an antagonist. Variants of the DGAT2 family memberproteins can be generated by mutagenesis, e.g., discrete point mutation,the insertion or deletion of sequences or the truncation of a DGAT2family member protein. An agonist of the DGAT2 family member proteinscan retain substantially the same, or a subset, of the biologicalactivities of the naturally occurring form of a DGAT2 family memberprotein (e.g., diacylglycerol acyltransferase activity). An antagonistof a DGAT2 family member protein can inhibit one or more of theactivities of the naturally occurring form of the DGAT2 family memberprotein by, for example, competitively modulating a DGAT2 familymember-mediated activity of a DGAT2 family member protein. Thus,specific biological effects can be elicited by treatment with a variantof limited function. Preferably, treatment of a subject with a varianthaving a subset of the biological activities of the naturally occurringform of the protein has fewer side effects in a subject relative totreatment with the naturally occurring form of the DGAT2 family memberprotein.

Variants of a DGAT2 family member protein can be identified by screeningcombinatorial libraries of mutants, e.g., truncation mutants, of a DGAT2family member protein for agonist or antagonist activity.

Libraries of fragments e.g., N terminal, C terminal, or internalfragments, of a DGAT2 family member protein coding sequence can be usedto generate a variegated population of fragments for screening andsubsequent selection of variants of a DGAT2 family member protein.

Variants in which a cysteine residues is added or deleted or in which aresidue which is glycosylated is added or deleted are particularlypreferred.

Methods for screening gene products of combinatorial libraries made bypoint mutations or truncation, and for screening cDNA libraries for geneproducts having a selected property. Recursive ensemble mutagenesis(REM), a technique which enhances the frequency of functional mutants inthe libraries, can be used in combination with the screening assays toidentify DGAT2 family member variants (Arkin and Yourvan, (1992) Proc.Natl. Acad. Sci. USA 89:7811-7815; Delgrave et al., (1993) ProteinEngineering 6(3):327-331).

Cell based assays can be exploited to analyze a variegated DGAT2 familymember library. For example, a library of expression vectors can betransfected into a cell line, e.g., a cell line, which ordinarilyresponds to DGAT2 family member in a substrate-dependent manner. Thetransfected cells are then contacted with DGAT2 family member and theeffect of the expression of the mutant on signaling by the DGAT2 familymember substrate can be detected, e.g., by measuring diacylglycerolacyltransferase activity. Plasmid DNA can then be recovered from thecells which score for inhibition, or alternatively, potentiation ofsignaling by the DGAT2 family member substrate, and the individualclones further characterized.

In another aspect, the invention features a method of making a DGAT2family member polypeptide, e.g., a peptide having a non-wild typeactivity, e.g., an antagonist, agonist, or super agonist of a naturallyoccurring DGAT2 family member polypeptide, e.g., a naturally occurringDGAT2 family member polypeptide. The method includes: altering thesequence of a DGAT2 family member polypeptide, e.g., altering thesequence, e.g., by substitution or deletion of one or more residues of anon-conserved region, a domain or residue disclosed herein, and testingthe altered polypeptide for the desired activity.

In another aspect, the invention features a method of making a fragmentor analog of a DGAT2 family member polypeptide having a biologicalactivity of a naturally occurring DGAT2 family member polypeptide. Themethod includes: altering the sequence, e.g., by substitution ordeletion of one or more residues, of a DGAT2 family member polypeptide,e.g., altering the sequence of a non-conserved region, or a domain orresidue described herein, and testing the altered polypeptide for thedesired activity.

Anti-DGAT2 Family Member Antibodies

In another aspect, the invention provides an anti-DGAT2 family memberantibody. The term “antibody” as used herein refers to an immunoglobulinmolecule or immunologically active portion thereof, i.e., anantigen-binding portion. Examples of immunologically active portions ofimmunoglobulin molecules include F(ab) and F(ab′)₂ fragments which canbe generated by treating the antibody with an enzyme such as pepsin.

The antibody can be a polyclonal, monoclonal, recombinant, e.g., achimeric or humanized, fully human, non-human, e.g., murine, or singlechain antibody. In a preferred embodiment it has effector function andcan fix complement. The antibody can be coupled to a toxin or imagingagent.

A full-length DGAT2 family member protein or, antigenic peptide fragmentof DGAT2 family member can be used as an immunogen or can be used toidentify anti-DGAT2 family member antibodies made with other immunogens,e.g., cells, membrane preparations, and the like. The antigenic peptideof DGAT2 family member should include at least 8 amino acid residues ofa DGAT2 family member amino acid sequence of the invention (e.g., theamino acid sequence shown in SEQ ID NO:8 or SEQ ID NO:20 or SEQ IDNO:62) and encompasses an epitope of DGAT2 family member. Preferably,the antigenic peptide includes at least 10 amino acid residues, morepreferably at least 15 amino acid residues, even more preferably atleast 20 amino acid residues, and most preferably at least 30 amino acidresidues.

Fragments of DGAT2 family member polypeptides of the invention can be,e.g., as immunogens, or used to characterize the specificity of anantibody or antibodies against what are believed to be hydrophilicregions of the DGAT2 family member protein. Similarly, a fragment ofDGAT2 family member proteins of the invention can be used to make anantibody against what is believed to be a hydrophobic region of theDGAT2 family member protein; a fragment of DGAT2 family can be used tomake an antibody against a diacylglycerol acyltransferase region of theDGAT2 family member protein.

Antibodies reactive with, or specific for, any of these regions, orother regions or domains described herein are provided.

In a preferred embodiment the antibody fails to bind an Fc receptor,e.g. it is a type which does not support Fc receptor binding or has beenmodified, e.g., by deletion or other mutation, such that is does nothave a functional Fc receptor binding region.

Preferred epitopes encompassed by the antigenic peptide are regions ofDGAT2 family member are located on the surface of the protein, e.g.,hydrophilic regions, as well as regions with high antigenicity. Forexample, an Emini surface probability analysis of the human DGAT2 familymember protein sequence can be used to indicate the regions that have aparticularly high probability of being localized to the surface of theDGAT2 family member protein and are thus likely to constitute surfaceresidues useful for targeting antibody production. Methods to determineEmini surface probability analysis or other methods to determineimmunogenic peptides of the DGAT2 family member amino acid sequences ofthe invention are known in the art.

In a preferred embodiment an antibody binds an epitope on any domain orregion of any of the DGAT2 family member proteins described herein.

Chimeric, humanized, but most preferably, completely human antibodiesare desirable for applications which include repeated administration,e.g., therapeutic treatment (and some diagnostic applications) of humanpatients.

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Such antibodies can be produced usingtransgenic mice that are incapable of expressing endogenousimmunoglobulin heavy and light chains genes, but which can express humanheavy and light chain genes. See, for example, Lonberg and Huszar (1995)Int. Rev. Immunol. 13:65-93); and U.S. Pat. Nos. 5,625,126; 5,633,425;5,569,825; 5,661,016; and 5,545,806. In addition, companies such asAbgenix, Inc. (Fremont, Calif.) and Medarex, Inc. (Princeton, N.J.), canbe engaged to provide human antibodies directed against a selectedantigen using technology similar to that described above.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a murineantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. This technology is described by Jespers etal. (1994) Bio/Technology 12:899-903).

The anti-DGAT2 family member antibody can be a single chain antibody. Asingle-chain antibody (scFV) may be engineered (see, for example,Colcher, D. et al., Ann. NY Acad. Sci. 1999 Jun. 30; 880:263-80; andReiter, Y., Clin. Cancer Res. 1996 Feb.; 2(2):245-52). The single chainantibody can be dimerized or multimerized to generate multivalentantibodies having specificities for different epitopes of the sametarget DGAT2 family member protein.

In a preferred embodiment, the antibody has reduced or no ability tobind an Fc receptor. For example, it is an isotype or subtype, fragmentor other mutant, which does not support binding to an Fc receptor, e.g.,it has a mutagenized or deleted Fc receptor binding region.

An anti-DGAT2 family member antibody (e.g., monoclonal antibody) can beused to isolate DGAT2 family member proteins or complexes by standardtechniques, such as affinity chromatography or immunoprecipitation.Moreover, an anti-DGAT2 family member antibody can be used to detectDGAT2 family member protein (e.g., in a cellular lysate or cellsupernatant) in order to evaluate the abundance and pattern ofexpression of the protein. Anti-DGAT2 family member antibodies can beused diagnostically to monitor protein levels in tissue as part of aclinical testing procedure, e.g., to, for example, determine theefficacy of a given treatment regimen. Detection can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance (i.e., antibody labeling). Examples of detectable substancesinclude various enzymes, prosthetic groups, fluorescent materials,luminescent materials, bioluminescent materials, and radioactivematerials. Examples of suitable enzymes include horseradish peroxidase,alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examplesof suitable prosthetic group complexes include streptavidin/biotin andavidin/biotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

An antibody (or fragment thereof) may be conjugated to a therapeuticmoiety such as a cytotoxin, a therapeutic agent or a radioactive ion. Acytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include taxol, cytochalasin B, gramicidin D, ethidiumbromide, emetine, mitomycin, etoposide, tenoposide, vincristine,vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone,glucocorticoids, procaine, tetracaine, lidocaine, propranolol,puromycin, maytansinoids, e.g., maytansinol (see U.S. Pat. No.5,208,020), CC-1065 (see U.S. Pat. Nos. 5,475,092, 5,585,499, 5,846,545)and analogs or homologs thereof. Therapeutic agents include, but are notlimited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine,6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylatingagents (e.g., mechlorethamine, thioepa chlorambucil, CC-1065, melphalan,carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan,dibromomannitol, streptozotocin, mitomycin C, and cis-dichlorodiamineplatinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin(formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin(formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)),and anti-mitotic agents (e.g., vincristine, vinblastine, taxol andmaytansinoids). Radioactive ions include, but are not limited to iodine,yttrium and praseodymium.

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic moiety is not to be construed aslimited to classical chemical therapeutic agents. For example, thetherapeutic moiety may be a protein or polypeptide possessing a desiredbiological activity. Such proteins may include, for example, a toxinsuch as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; aprotein such as tumor necrosis factor, α-interferon, β-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Recombinant Expression Vectors, Host Cells and Genetically EngineeredCells

In another aspect, the invention includes, vectors, preferablyexpression vectors, containing a nucleic acid encoding a polypeptidedescribed herein. As used herein, the term “vector” refers to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked and can include a plasmid, cosmid or viral vector. Thevector can be capable of autonomous replication or it can integrate intoa host DNA. Viral vectors include, e.g., replication defectiveretroviruses, adenoviruses and adeno-associated viruses.

A vector can include a DGAT2 family member nucleic acid of the inventionin a form suitable for expression of the nucleic acid in a host cell.Preferably the recombinant expression vector includes one or moreregulatory sequences operatively linked to the nucleic acid sequence tobe expressed. The term “regulatory sequence” includes promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Regulatory sequences include those which direct constitutiveexpression of a nucleotide sequence, as well as tissue-specificregulatory and/or inducible sequences. The design of the expressionvector can depend on such factors as the choice of the host cell to betransformed, the level of expression of protein desired, and the like.The expression vectors of the invention can be introduced into hostcells to thereby produce proteins or polypeptides, including fusionproteins or polypeptides, encoded by nucleic acids as described herein(e.g., DGAT2 family member proteins, mutant forms of DGAT2 family memberproteins, fusion proteins, and the like).

The recombinant expression vectors of the invention can be designed forexpression of DGAT2 family member proteins in prokaryotic or eukaryoticcells. For example, polypeptides of the invention can be expressed in E.coli, insect cells (e.g., using baculovirus expression vectors), yeastcells or mammalian cells. Suitable host cells are discussed further inGoeddel, Gene Expression Technology: Methods in Enzymology 185, AcademicPress, San Diego, Calif. (1990). Alternatively, the recombinantexpression vector can be transcribed and translated in vitro, forexample using T7 promoter regulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out in E.coli with vectors containing constitutive or inducible promotersdirecting the expression of either fusion or non-fusion proteins. Fusionvectors add a number of amino acids to a protein encoded therein,usually to the amino terminus of the recombinant protein. Such fusionvectors typically serve three purposes: 1) to increase expression ofrecombinant protein; 2) to increase the solubility of the recombinantprotein; and 3) to aid in the purification of the recombinant protein byacting as a ligand in affinity purification. Often, a proteolyticcleavage site is introduced at the junction of the fusion moiety and therecombinant protein to enable separation of the recombinant protein fromthe fusion moiety subsequent to purification of the fusion protein. Suchenzymes, and their cognate recognition sequences, include Factor Xa,thrombin and enterokinase. Typical fusion expression vectors includepGEX (Pharmacia Biotech Inc; Smith, D. B. and Johnson, K. S., (1988)Gene 67:31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5(Pharmacia, Piscataway, N.J.) which fuse glutathione S-transferase(GST), maltose E binding protein, or protein A, respectively, to thetarget recombinant protein.

Purified fusion proteins can be used in DGAT2 family member activityassays, (e.g., direct assays or competitive assays described in detailbelow), or to generate antibodies specific for DGAT2 family memberprotein(s). In a preferred embodiment, a fusion protein expressed in aretroviral expression vector of the present invention can be used toinfect bone marrow cells which are subsequently transplanted intoirradiated recipients. The pathology of the subject recipient is thenexamined after sufficient time has passed (e.g., six (6) weeks).

To maximize recombinant protein expression in E. coli is to express theprotein in host bacteria with an impaired capacity to proteolyticallycleave the recombinant protein (Gottesman, S., Gene ExpressionTechnology: Methods in Enzymology 185, Academic Press, San Diego, Calif.(1990) 119-128). Another strategy is to alter the nucleic acid sequenceof the nucleic acid to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin E. coli (Wada et al., (1992) Nucleic Acids Res. 20:2111-2118). Suchalteration of nucleic acid sequences of the invention can be carried outby standard DNA synthesis techniques.

The DGAT2 family member expression vector can be a yeast expressionvector, a vector for expression in insect cells, e.g., a baculovirusexpression vector or a vector suitable for expression in mammaliancells.

When used in mammalian cells, the expression vector's control functionsare often provided by viral regulatory elements. For example, commonlyused promoters are derived from polyoma, Adenovirus 2, cytomegalovirusand Simian Virus 40.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Non-limiting examples of suitabletissue-specific promoters include the albumin promoter (liver-specific;Pinkert et al., (1987) Genes Dev. 1:268-277), lymphoid-specificpromoters (Calame and Eaton, (1988) Adv. Immunol. 43:235-275), inparticular promoters of T cell receptors (Winoto and Baltimore, (1989)EMBO J. 8:729-733) and immunoglobulins (Banerji et al., (1983) Cell33:729-740; Queen and Baltimore, (1983) Cell 33:741-748),neuron-specific promoters (e.g., the neurofilament promoter; Byrne andRuddle, (1989) Proc. Natl. Acad. Sci. USA 86:5473-5477),pancreas-specific promoters (Edlund et al., (1985) Science 230:912-916),and mammary gland-specific promoters (e.g., milk whey promoter; U.S.Pat. No. 4,873,316 and European Application Publication No. 264,166).Developmentally-regulated promoters are also encompassed, for example,the murine hox promoters (Kessel and Gruss, (1990) Science 249:374-379)and the α-fetoprotein promoter (Campes and Tilghman, (1989) Genes Dev.3:537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule of the invention cloned into the expressionvector in an antisense orientation. Regulatory sequences (e.g., viralpromoters and/or enhancers) operatively linked to a nucleic acid clonedin the antisense orientation can be chosen which direct theconstitutive, tissue specific or cell type specific expression ofantisense RNA in a variety of cell types. The antisense expressionvector can be in the form of a recombinant plasmid, phagemid orattenuated virus. For a discussion of the regulation of gene expressionusing antisense genes see Weintraub, H. et al., Antisense RNA as amolecular tool for genetic analysis, Reviews—Trends in Genetics, Vol.1(1) 1986.

Another aspect the invention provides a host cell which includes anucleic acid molecule described herein, e.g., a DGAT2 family membernucleic acid molecule within a recombinant expression vector or a DGAT2family member nucleic acid molecule containing sequences which allow itto homologously recombine into a specific site of the host cell'sgenome. The terms “host cell” and “recombinant host cell” are usedinterchangeably herein. Such terms refer not only to the particularsubject cell but rather also to the progeny or potential progeny of sucha cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, aDGAT2 family member protein can be expressed in bacterial cells such asE. coli, insect cells, yeast or mammalian cells (such as Chinese hamsterovary cells (CHO) or COS cells). Other suitable host cells are known tothose skilled in the art.

Vector DNA can be introduced into host cells via conventionaltransformation or transfection techniques. As used herein, the terms“transformation” and “transfection” are intended to refer to a varietyof art-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation

A host cell of the invention can be used to produce (i.e., express) aDGAT2 family member protein. Accordingly, the invention further providesmethods for producing a DGAT2 family member protein using the host cellsof the invention. In one embodiment, the method includes culturing thehost cell of the invention (into which a recombinant expression vectorencoding a DGAT2 family member protein has been introduced) in asuitable medium such that a DGAT2 family member protein is produced. Inanother embodiment, the method further includes isolating a DGAT2 familymember protein from the medium or the host cell.

In another aspect, the invention features, a cell or purifiedpreparation of cells which include a DGAT2 family member transgene, orwhich otherwise misexpress one or more DGAT2 family member molecules.The cell preparation can consist of human or non-human cells, e.g.,rodent cells, e.g., mouse or rat cells, rabbit cells, or pig cells. Inpreferred embodiments, the cell or cells include a DGAT2 family membertransgene, e.g., a heterologous form of a DGAT2 family member, e.g., agene derived from humans (in the case of a non-human cell). The DGAT2family member transgene can be misexpressed, e.g., overexpressed orunderexpressed. In other preferred embodiments, the cell or cellsinclude a gene which misexpress an endogenous DGAT2 family member, e.g.,a gene the expression of which is disrupted, e.g., a knockout. Suchcells can serve as a model for studying disorders which are related tomutated or mis-expressed DGAT2 family member alleles or for use in drugscreening.

In another aspect, the invention features, a human cell, e.g., ahematopoietic stem cell, transformed with nucleic acid which encodes asubject DGAT2 family member polypeptide.

Also provided are cells or a purified preparation thereof, e.g., humancells, in which an endogenous DGAT2 family member is under the controlof a regulatory sequence that does not normally control the expressionof the endogenous DGAT2 family member gene. The expressioncharacteristics of an endogenous gene within a cell, e.g., a cell lineor microorganism, can be modified by inserting a heterologous DNAregulatory element into the genome of the cell such that the insertedregulatory element is operably linked to the endogenous DGAT2 familymember gene. For example, an endogenous DGAT2 family member gene, e.g.,a gene which is “transcriptionally silent,” e.g., not normallyexpressed, or expressed only at very low levels, may be activated byinserting a regulatory element which is capable of promoting theexpression of a normally expressed gene product in that cell. Techniquessuch as targeted homologous recombinations, can be used to insert theheterologous DNA as described in, e.g., Chappel, U.S. Pat. No.5,272,071; WO 91/06667, published on May 16, 1991.

Transgenic Animals

The invention provides non-human transgenic animals. Such animals areuseful for studying the function and/or activity of a DGAT2 familymember protein and for identifying and/or evaluating modulators of DGAT2family member activity. As used herein, a “transgenic animal” is anon-human animal, preferably a mammal, more preferably a rodent such asa rat or mouse, in which one or more of the cells of the animal includesa transgene. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, amphibians, and the like.A transgene is exogenous DNA or a rearrangement, e.g., a deletion ofendogenous chromosomal DNA, which preferably is integrated into oroccurs in the genome of the cells of a transgenic animal. A transgenecan direct the expression of an encoded gene product in one or more celltypes or tissues of the transgenic animal, other transgenes, e.g., aknockout, reduce expression. Thus, a transgenic animal can be one inwhich an endogenous DGAT2 family member gene has been altered by, e.g.,by homologous recombination between the endogenous gene and an exogenousDNA molecule introduced into a cell of the animal, e.g., an embryoniccell of the animal, prior to development of the animal.

Intronic sequences and polyadenylation signals can also be included inthe transgene to increase the efficiency of expression of the transgene.A tissue-specific regulatory sequence(s) can be operably linked to atransgene of the invention to direct expression of a DGAT2 family memberprotein to particular cells. A transgenic founder animal can beidentified based upon the presence of a DGAT2 family member transgene inits genome and/or expression of DGAT2 family member mRNA in tissues orcells of the animals. A transgenic founder animal can then be used tobreed additional animals carrying the transgene. Moreover, transgenicanimals carrying a transgene encoding a DGAT2 family member protein canfurther be bred to other transgenic animals carrying other transgenes.

DGAT2 family member proteins or polypeptides can be expressed intransgenic animals or plants, e.g., a nucleic acid encoding the proteinor polypeptide can be introduced into the genome of an animal. Inpreferred embodiments the nucleic acid is placed under the control of atissue specific promoter, e.g., a milk or egg specific promoter, andrecovered from the milk or eggs produced by the animal. Suitable animalsare mice, pigs, cows, goats, and sheep.

The invention also includes a population of cells from a transgenicanimal, as discussed herein.

Uses

The nucleic acid molecules, proteins, protein homologues, and antibodiesdescribed herein can be used in one or more of the following methods: a)screening assays; b) predictive medicine (e.g., diagnostic assays,prognostic assays, monitoring clinical trials, and pharmacogenetics);and c) methods of treatment (e.g., therapeutic and prophylactic). Inparticularly preferred embodiments, the compositions provided herein areused in conjunction with methods of diagnosis and treatment of metabolicdisorders (e.g., obesity, hyperlipidemia, diabetes), as well ascardiovascular and liver disorders.

The isolated nucleic acid molecules of the invention can be used, forexample, to express a DGAT2 family member protein (e.g., via arecombinant expression vector in a host cell in gene therapyapplications), to detect a DGAT2 family member mRNA (e.g., in abiological sample such as adipose tissue) or a genetic alteration in aDGAT2 family member gene, and to modulate DGAT2 family member activity,as described further below. The DGAT2 family member proteins can be usedto treat disorders characterized by insufficient or excessive productionof a DGAT2 family member substrate or production of DGAT2 family memberinhibitors (e.g., an obesity disorder). In addition, the DGAT2 familymember proteins can be used to screen for naturally occurring DGAT2family member substrates, to screen for drugs or compounds whichmodulate DGAT2 family member activity, as well as to treat disorderscharacterized by insufficient or excessive production of DGAT2 familymember protein or production of DGAT2 family member protein forms whichhave decreased, aberrant or unwanted activity compared to DGAT2 familymember wild-type protein. Such disorders include those characterized byaberrant signaling or aberrant, e.g., hyperproliferative, cell growth.Moreover, the anti-DGAT2 family member antibodies of the invention canbe used to detect and isolate DGAT2 family member proteins, regulate thebioavailability of DGAT2 family member proteins, and modulate DGAT2family member activity.

A method of evaluating a compound for the ability to interact with,e.g., bind, a subject DGAT2 family member polypeptide is provided. Themethod includes: contacting the compound with the subject DGAT2 familymember polypeptide; and evaluating ability of the compound to interactwith, e.g., to bind or form a complex with the subject DGAT2 familymember polypeptide. This method can be performed in vitro, e.g., in acell free system, or in vivo, e.g., in a two-hybrid interaction trapassay. This method can be used to identify naturally occurring moleculeswhich interact with subject DGAT2 family member polypeptide. It can alsobe used to find natural or synthetic inhibitors of subject DGAT2 familymember polypeptide. Screening methods are discussed in more detailbelow.

Screening Assays:

The invention provides methods (also referred to herein as “screeningassays”) for identifying modulators, i.e., candidate or test compoundsor agents (e.g., proteins, peptides, peptidomimetics, peptoids, smallmolecules or other drugs) which bind to DGAT2 family member proteins,have a stimulatory or inhibitory effect on, for example, DGAT2 familymember expression or DGAT2 family member activity, or have a stimulatoryor inhibitory effect on, for example, the expression or activity of aDGAT2 family member substrate. Compounds thus identified can be used tomodulate the activity of target gene products (e.g., DGAT2 family membergenes) in a therapeutic protocol, to elaborate the biological functionof the target gene product, or to identify compounds that disrupt normaltarget gene interactions.

The enzyme reaction catalyzed by (DGATs) involves the coupling of anacyl-CoA to a preformed diacylglycerol producing one equivalent ofCoenzyme A (CoA) and triacylglycerol. Assays for DGAT activity are knownin the art and can include, but are not limited to, direct detection ofthe products (Coenzyme A or triacylglycerol) or detection in theconsumption of the substrates (diacylglycerol or acyl-CoA). PreviousDGAT assays have focused on generation of a radiolabeled triacylglycerolusing either a radiolabled diacylglycerol or acyl-CoA starting material.(Lardizabal, K. K., Mai, J. T., Wagner, N. W., Wyrick, A., Voelker, T.,and Hawkins, D. J. J. Biol. Chem. 276 (2001) 38862-38869; Cases, S.,Stone, S. J., Zhou, P., Yen, E., Tow, B., Lardizabal, K. D., Voelker,T., and Farese Jr., R. V. J. Biol. Chem. 276 (2001) 38870-38876). Thisis a laborious procedure involving organic extractions and separationsthat are not rigorously quantitative for accurate kineticcharacterization of the enzyme. This procedure can be extended to a morequantitative assay wherein an aqueous reaction with radiolabeledsubstrate (either acyl-CoA or diacylglycerol) is followed by separationand detection using radiometric HPLC. This will allow for separation anddetection of the various reaction components (as TLC does) but allow foraccurate quantitation of the various reaction species. However, thisapproach is not amenable to high-throughput screening.

Matrix-assisted laser desorption/ionization time-of-flight massspectrometry (MALDI-TOF) and liquid chromatography/mass spectrometry(LC/MS) are very sensitive techniques that do not require the use ofradiolabled substrates. This has been previously employed for detectionof triacylglycerols. (Hlongwane, C., Delves, I. G., Wan, L. W., andAyorinde, F. O. Rapid Commun. Mass Spectrom. 15 (2001) 2027-2034;Ayorinde, F. O., Keith Jr. Q. L., and Wan, L. W. Rapid Communi. MassSpectrom. 13 (199) 1762-1769; Byrdwell, W. C., Emken, E. A., Neff, W.E., Adlof, R. O. Lipids. 31 (1996) 919-935). These and relatedtechniques will allow for quantitation of every component in the DGATreaction.

In one aspect, a high-throughput assay for monitoring the DGAT assayrelies on detection of the free thiol generated in the form of CoA.Dithiobis-(2-nitro-5-thiobenzoic acid) (DTNB) has been employed formonitoring the reaction of numerous acyltransferases includingmonoacylglycerol acyltransferases (MGATs). (Bierbach, H. Digestion. 28(1983) 138-147). Alternatively, fluorescent thiol substrates may beutilized in assays and detected using standard fluorescent detectionmethods known in the art. One example of detection includes usingThioGlo (NovaBiochem). Storey, B. T., et al. 1998. Mol. Reprod. Dev. 49,400; Wright, S. K., and Viola, R. E. 1998. Anal. Biochem. 265, 8; andLangmuir, M. E., et al. 1996. in Fluorescence Microscopy and FluorescentProbes (Slavic, J., ed.) pp. 229-233, Plenum Press, New York.

In yet another aspect, a high throughput assay for monitoring the DGATassay relies on detection of product generated using fluorescenceresonance energy transfer (FRET) analysis. In this method, substrates(acyl coA and diacylglycerol) are each measured with an appropriatefluorophore. Formation of the resulting triglyceride may be monitoredusing standard FRET analysis procedures. See, e.g., Stryer L, Haugland RP. Proc Natl Acad Sci USA 58, 719-726 (1967); and Selvin P R. MethodsEnzymol 246, 300-334 (1995).

These approaches would be amenable to high-throughput screening as wellas continuous assays for kinetic characterization and determination ofinhibitory activity by small molecule inhibitors.

In another aspect, a high-throughput assay for monitoring the DGAT assayrelies on detection of triacylglycerol product generated as a result ofDGAT enzyme activity. In this method, scintillation proximity assay(SPA) technology may be utilized to monitor the acyltransferasereaction. In this method, one substrate is biotinylated (e.g., abiotinylated fatty-acyl-CoA) and combined in the reaction withradiolabeled second substrate (e.g., radiolabeled diacylglyceride, e.g.,Diolein). In one aspect the biotinylated substrate can be a donor fattyacyl coA, and the radiolabeled substrate can be a radiolabeled acceptordiacylglycerol. In another aspect, the biotinylated substrate can be abiotinylated acceptor diacylglycerol and the radiolabeled secondsubstrate can be a radiolabeled donor fatty acyl coA. Either combinationmay be used, and optimized to suit conditions. Either combination ofsubstrates result in generation of a biotinylated, radiolabeled producttriacylglycerol. Upon completion of the enzyme assay, producttriacylglycerol generation can be determined using standard techniquesfor collection of biotinylated product and detection of fluorescence(e.g., SPA technology; avidin coated plates and traditional radiometricdetection). The SPA beads are a preferred method of detection in manyinstances, as the SPA bead (Amersham) has both avidin and a scintillantcovalently attached such that when radiolabeled biotinylated substrateattaches to the beads, the isotope is already in close proximity to thescintillant, thus making the addition of scintillation fluidunnecessary. This also means that only those molecules bound to thebeads represent the radioactivity of the resulting product.

As described above, this assay can be utilized to monitor anacyltransferase reaction where either the donor acyl-CoA is biotinylatedand the acceptor is radiolabeled; or a reaction where the donor isradiolabeled and the acceptor is biotinylated. Thus, the present assaymay be useful to monitor any acyltransferase activity in whichsubstrates are amenable to labeling in a similar manner. Thus thepresent assay is applicable to each of the DGAT2 family membersdescribed herein, and may also be applied to other acyltransferaseenzymes (e.g., DGAT1). The present assay has advantages over artrecognized methods of detection: the product can be captured throughutilization of the biotin label (e.g., on a SPA bead) rather thanlaborious organic extractions; radiolabel sensitivity assay is muchhigher than detection of the fluorescent free CoA released; and thepresent methods are adaptable to high throughput screening as well ascontinuous assays for kinetic characterization and determination ofinhibitory activity by small molecule inhibitors. Examples of thesubstrates and product reaction include:

In one embodiment, the invention provides assays for screening candidateor test compounds which are substrates of a DGAT2 family member proteinor polypeptide or a biologically active portion thereof. In anotherembodiment, the invention provides assays for screening candidate ortest compounds which bind to or modulate the activity of a DGAT2 familymember protein or polypeptide or a biologically active portion thereof.

The test compounds of the present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in theart, including: biological libraries; peptoid libraries [libraries ofmolecules having the functionalities of peptides, but with a novel,non-peptide backbone which are resistant to enzymatic degradation butwhich nevertheless remain bioactive] (see, e.g., Zuckermann, R. N. etal., J. Med. Chem. 1994, 37: 2678-85); spatially addressable parallelsolid phase or solution phase libraries; synthetic library methodsrequiring deconvolution; the ‘one-bead one-compound’ library method; andsynthetic library methods using affinity chromatography selection. Thebiological library and peptoid library approaches are limited to peptidelibraries, while the other four approaches are applicable to peptide,non-peptide oligomer or small molecule libraries of compounds (Lam, K.S. (1997) Anticancer Drug Des. 12:145).

Examples of methods for the synthesis of molecular libraries can befound in the art, for example in: DeWitt et al. (1993) Proc. Natl. Acad.Sci. U.S.A. 90:6909; Erb et al., (1994) Proc. Natl. Acad. Sci. USA91:11422; Zuckermann et al., (1994). J. Med. Chem. 37:2678; Cho et al.,(1993) Science 261:1303; Carrell et al., (1994) Angew. Chem. Int. Ed.Engl. 33:2059; Carell et al., (1994) Angew. Chem. Int. Ed. Engl.33:2061; and in Gallop et al., (1994) J. Med. Chem. 37:1233.

Libraries of compounds may be presented in solution (e.g., Houghten,(1992) Biotechniques 13:412-421), or on beads (Lam, (1991) Nature354:82-84), chips (Fodor, (1993) Nature 364:555-556), bacteria or spores(Ladner, U.S. Pat. No. 5,223,409), plasmids (Cull et al., (1992) Proc.Natl. Acad. Sci. USA 89:1865-1869) or on phage (Scott and Smith, (1990)Science 249:386-390); (Devlin, (1990) Science 249:404-406); (Cwirla etal., (1990) Proc. Natl. Acad. Sci. 87:6378-6382); (Felici, (1991) J.Mol. Biol. 222:301-310); (Ladner supra.).

Preferred libraries of compounds for the screening methods of theinvention include small molecule compounds based on natural substratesfor the DGAT2 family members of the invention (e.g., acyl-CoA,diacylglycerol). Generation of small molecules and analogs based on thesubstrates can be produced using methods described in the referencescited above, in combination with additional methods and skills known toone in the art.

In one embodiment, an assay is a cell-based assay in which a cell whichexpresses a DGAT2 family member protein or biologically active portionthereof is contacted with a test compound, and the ability of the testcompound to modulate DGAT2 family member activity is determined.Determining the ability of the test compound to modulate DGAT2 familymember activity can be accomplished by monitoring, for example,diacylglycerol acyltransferase activity. The cell, for example, can beof mammalian origin, e.g., human. Cell homogenates, or fractions,preferably membrane containing fractions, including microsomes, can alsobe tested.

The ability of the test compound to modulate DGAT2 family member bindingto a compound, e.g., a DGAT2 family member substrate, or to bind toDGAT2 family member can also be evaluated. This can be accomplished, forexample, by coupling the compound, e.g., the substrate, with aradioisotope or enzymatic label such that binding of the compound, e.g.,the substrate, to DGAT2 family member can be determined by detecting thelabeled compound, e.g., substrate, in a complex. Alternatively, DGAT2family member could be coupled with a radioisotope or enzymatic label tomonitor the ability of a test compound to modulate DGAT2 family memberbinding to a DGAT2 family member substrate in a complex. For example,compounds (e.g., DGAT2 family member substrates) can be labeled with¹²⁵I, ³⁵S, ¹⁴C, or ³H, either directly or indirectly, and theradioisotope detected by direct counting of radioemmission or byscintillation counting. Alternatively, compounds can be enzymaticallylabeled with, for example, horseradish peroxidase, alkaline phosphatase,or luciferase, and the enzymatic label detected by determination ofconversion of an appropriate substrate to product.

The ability of a compound (e.g., a DGAT2 family member substrate) tointeract with DGAT2 family member with or without the labeling of any ofthe interactants can be evaluated. For example, interaction of acompound with DGAT2 family member without the labeling of either thecompound or the DGAT2 family member can be measured by the change in theamount of triacylglycerol synthesis in response to contact of acompound. Changes in this triacylglycerol synthesis rate can be used asan indicator of the interaction between a compound and DGAT2 familymember.

In yet another embodiment, a cell-free assay is provided in which aDGAT2 family member protein or biologically active portion thereof iscontacted with a test compound and the ability of the test compound tobind to the DGAT2 family member protein or biologically active portionthereof is evaluated. Preferred biologically active portions of theDGAT2 family member proteins to be used in assays of the presentinvention include fragments which participate in interactions withnon-DGAT2 family member molecules, e.g., fragments with high surfaceprobability scores, fragments which interact with substrates of DGAT2family members.

Soluble and/or membrane-bound forms of isolated proteins (e.g., DGAT2family member proteins or biologically active portions thereof) can beused in the cell-free assays of the invention. When membrane-bound formsof the protein are used, it may be desirable to utilize a solubilizingagent. Examples of such solubilizing agents include non-ionic detergentssuch as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside,octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, Triton® X-100,Triton® X-114, Thesit®, Isotridecypoly(ethylene glycol ether)_(n),3-[(3-cholamidopropyl)dimethylamminio]-1-propane sulfonate (CHAPS),3-[(3-cholamidopropyl)dimethylamminio]-2-hydroxy-1-propane sulfonate(CHAPSO), or N-dodecyl-N,N-dimethyl-3-ammonio-1-propane sulfonate.

Cell-free assays involve preparing a reaction mixture of the target geneprotein and the test compound under conditions and for a time sufficientto allow the two components to interact and bind, thus forming a complexthat can be removed and/or detected.

In one embodiment, assays are performed where the ability of an agent toblock diacylglycerol acyltransferase activity within a cell isevaluated.

The interaction between two molecules can also be detected, e.g., usingfluorescence energy transfer (FET) (see, for example, Lakowicz et al.,U.S. Pat. No. 5,631,169; Stavrianopoulos, et al., U.S. Pat. No.4,868,103). A fluorophore label on the first, ‘donor’ molecule isselected such that its emitted fluorescent energy will be absorbed by afluorescent label on a second, ‘acceptor’ molecule, which in turn isable to fluoresce due to the absorbed energy. Alternately, the ‘donor’protein molecule may simply utilize the natural fluorescent energy oftryptophan residues. Labels are chosen that emit different wavelengthsof light, such that the ‘acceptor’ molecule label may be differentiatedfrom that of the ‘donor’. Since the efficiency of energy transferbetween the labels is related to the distance separating the molecules,the spatial relationship between the molecules can be assessed. In asituation in which binding occurs between the molecules, the fluorescentemission of the ‘acceptor’ molecule label in the assay should bemaximal. An FET binding event can be conveniently measured throughstandard fluorometric detection means well known in the art (e.g., usinga fluorimeter).

In another embodiment, determining the ability of the DGAT2 familymember protein to bind to a target molecule can be accomplished usingreal-time Biomolecular Interaction Analysis (BIA) (see, e.g., Sjolander,S. and Urbaniczky, C., (1991) Anal. Chem. 63:2338-2345 and Szabo et al.,(1995) Curr. Opin. Struct. Biol. 5:699-705). “Surface plasmon resonance”or “BIA” detects biospecific interactions in real time, without labelingany of the interactants (e.g., BIAcore). Changes in the mass at thebinding surface (indicative of a binding event) result in alterations ofthe refractive index of light near the surface (the optical phenomenonof surface plasmon resonance (SPR)), resulting in a detectable signalwhich can be used as an indication of real-time reactions betweenbiological molecules.

In one embodiment, the target gene product or the test substance isanchored onto a solid phase. The target gene product/test compoundcomplexes anchored on the solid phase can be detected at the end of thereaction. Preferably, the target gene product can be anchored onto asolid surface, and the test compound, (which is not anchored), can belabeled, either directly or indirectly, with detectable labels discussedherein.

It may be desirable to immobilize either DGAT2 family member, ananti-DGAT2 family member antibody or its target molecule to facilitateseparation of complexed from uncomplexed forms of one or both of theproteins, as well as to accommodate automation of the assay. Binding ofa test compound to a DGAT2 family member protein, or interaction of aDGAT2 family member protein with a target molecule in the presence andabsence of a candidate compound, can be accomplished in any vesselsuitable for containing the reactants. Examples of such vessels includemicrotiter plates, test tubes, and micro-centrifuge tubes. In oneembodiment, a fusion protein can be provided which adds a domain thatallows one or both of the proteins to be bound to a matrix. For example,glutathione-S-transferase/DGAT2 family member fusion proteins orglutathione-S-transferase/target fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtiter plates, which are then combined withthe test compound or the test compound and either the non-adsorbedtarget protein or DGAT2 family member protein, and the mixture incubatedunder conditions conducive to complex formation (e.g., at physiologicalconditions for salt and pH). Following incubation, the beads ormicrotiter plate wells are washed to remove any unbound components, thematrix immobilized in the case of beads, complex determined eitherdirectly or indirectly, for example, as described above. Alternatively,the complexes can be dissociated from the matrix, and the level of DGAT2family member binding or activity determined using standard techniques.

Other techniques for immobilizing either a DGAT2 family member proteinor a target molecule on matrices include using conjugation of biotin andstreptavidin. Biotinylated DGAT2 family member protein or targetmolecules can be prepared from biotin-NHS (N-hydroxy-succinimide) usingtechniques known in the art (e.g., biotinylation kit, Pierce Chemicals,Rockford, Ill.), and immobilized in the wells of streptavidin-coated 96well plates (Pierce Chemical).

In order to conduct the assay, the non-immobilized component is added tothe coated surface containing the anchored component. After the reactionis complete, unreacted components are removed (e.g., by washing) underconditions such that any complexes formed will remain immobilized on thesolid surface. The detection of complexes anchored on the solid surfacecan be accomplished in a number of ways. Where the previouslynon-immobilized component is pre-labeled, the detection of labelimmobilized on the surface indicates that complexes were formed. Wherethe previously non-immobilized component is not pre-labeled, an indirectlabel can be used to detect complexes anchored on the surface; e.g.,using a labeled antibody specific for the immobilized component (theantibody, in turn, can be directly labeled or indirectly labeled with,e.g., a labeled anti-Ig antibody).

In one embodiment, this assay is performed utilizing antibodies reactivewith DGAT2 family member protein or target molecules but which do notinterfere with binding of the DGAT2 family member protein to its targetmolecule. Such antibodies can be derivatized to the wells of the plate,and unbound target or DGAT2 family member protein trapped in the wellsby antibody conjugation. Methods for detecting such complexes, inaddition to those described above for the GST-immobilized complexes,include immunodetection of complexes using antibodies reactive with theDGAT2 family member protein or target molecule, as well as enzyme-linkedassays which rely on detecting an enzymatic activity associated with theDGAT2 family member protein or target molecule.

Alternatively, cell free assays can be conducted in a liquid phase. Insuch an assay, the reaction products are separated from unreactedcomponents, by any of a number of standard techniques, including but notlimited to: differential centrifugation (see, for example, Rivas, G.,and Minton, A. P., Trends Biochem Sci 1993 Aug.; 18(8):284-7);chromatography (gel filtration chromatography, ion-exchangechromatography); electrophoresis (see, e.g., Ausubel, F. et al., eds.Current Protocols in Molecular Biology 1999, J. Wiley: New York.); andimmunoprecipitation (see, for example, Ausubel, F. et al., eds. CurrentProtocols in Molecular Biology 1999, J. Wiley: New York). Such resinsand chromatographic techniques are known to one skilled in the art (see,e.g., Heegaard, N. H., J. Mol. Recognit. 1998 Winter; 11(16):141-8;Hage, D. S., and Tweed, S. A., J. Chromatogr. B Biomed. Sci. Appl. 1997Oct. 10; 699(1-2):499-525). Further, fluorescence energy transfer mayalso be conveniently utilized, as described herein, to detect bindingwithout further purification of the complex from solution.

In a preferred embodiment, the assay includes contacting the DGAT2family member protein or biologically active portion thereof with aknown compound which binds DGAT2 family member (e.g., substrate, e.g.,acyl-coA, diacylglycerol) to form an assay mixture, contacting the assaymixture with a test compound, and determining the ability of the testcompound to interact with a DGAT2 family member protein, whereindetermining the ability of the test compound to interact with a DGAT2family member protein includes determining the ability of the testcompound to preferentially bind to DGAT2 family member or biologicallyactive portion thereof, or to modulate the activity of a targetmolecule, as compared to the known compound (e.g., acyl-coA,diacylglycerol).

The target gene products of the invention can, in vivo, interact withone or more cellular or extracellular macromolecules, such as proteins.For the purposes of this discussion, such cellular and extracellularmacromolecules are referred to herein as “binding partners.” Compoundsthat disrupt such interactions can be useful in regulating the activityof the target gene product. Such compounds can include, but are notlimited to molecules such as antibodies, peptides, and small molecules.The preferred target genes/products for use in this embodiment are theDGAT2 family member genes herein identified. In an alternativeembodiment, the invention provides methods for determining the abilityof the test compound to modulate the activity of a DGAT2 family memberprotein through modulation of the activity of a downstream effector of aDGAT2 family member target molecule. For example, the activity of theeffector molecule on an appropriate target can be determined, or thebinding of the effector to an appropriate target can be determined, aspreviously described.

To identify compounds that interfere with the interaction between thetarget gene product and its cellular or extracellular bindingpartner(s), e.g., a substrate, e.g., acyl-coA, diacylglycerol, areaction mixture containing the target gene product and the bindingpartner is prepared, under conditions and for a time sufficient, toallow the two products to form complex. In order to test an inhibitoryagent, the reaction mixture is provided in the presence and absence ofthe test compound. The test compound can be initially included in thereaction mixture, or can be added at a time subsequent to the additionof the target gene and its cellular or extracellular binding partner.Control reaction mixtures are incubated without the test compound orwith a placebo. The formation of any complexes between the target geneproduct and the cellular or extracellular binding partner is thendetected. The formation of a complex in the control reaction, but not inthe reaction mixture containing the test compound, indicates that thecompound interferes with the interaction of the target gene product andthe interactive binding partner. Additionally, complex formation withinreaction mixtures containing the test compound and normal target geneproduct can also be compared to complex formation within reactionmixtures containing the test compound and mutant target gene product.This comparison can be important in those cases wherein it is desirableto identify compounds that disrupt interactions of mutant but not normaltarget gene products.

These assays can be conducted in a heterogeneous or homogeneous format.Heterogeneous assays involve anchoring either the target gene product orthe binding partner onto a solid phase, and detecting complexes anchoredon the solid phase at the end of the reaction. In homogeneous assays,the entire reaction is carried out in a liquid phase. In eitherapproach, the order of addition of reactants can be varied to obtaindifferent information about the compounds being tested. For example,test compounds that interfere with the interaction between the targetgene products and the binding partners, e.g., by competition, can beidentified by conducting the reaction in the presence of the testsubstance. Alternatively, test compounds that disrupt preformedcomplexes, e.g., compounds with higher binding constants that displaceone of the components from the complex, can be tested by adding the testcompound to the reaction mixture after complexes have been formed. Thevarious formats are briefly described below.

In a heterogeneous assay system, either the target gene product or theinteractive cellular or extracellular binding partner, is anchored ontoa solid surface (e.g., a microtiter plate), while the non-anchoredspecies is labeled, either directly or indirectly. The anchored speciescan be immobilized by non-covalent or covalent attachments.Alternatively, an immobilized antibody specific for the species to beanchored can be used to anchor the species to the solid surface.

In order to conduct the assay, the partner of the immobilized species isexposed to the coated surface with or without the test compound. Afterthe reaction is complete, unreacted components are removed (e.g., bywashing) and any complexes formed will remain immobilized on the solidsurface. Where the non-immobilized species is pre-labeled, the detectionof label immobilized on the surface indicates that complexes wereformed. Where the non-immobilized species is not pre-labeled, anindirect label can be used to detect complexes anchored on the surface;e.g., using a labeled antibody specific for the initiallynon-immobilized species (the antibody, in turn, can be directly labeledor indirectly labeled with, e.g., a labeled anti-Ig antibody). Dependingupon the order of addition of reaction components, test compounds thatinhibit complex formation or that disrupt preformed complexes can bedetected.

Alternatively, the reaction can be conducted in a liquid phase in thepresence or absence of the test compound, the reaction productsseparated from unreacted components, and complexes detected; e.g., usingan immobilized antibody specific for one of the binding components toanchor any complexes formed in solution, and a labeled antibody specificfor the other partner to detect anchored complexes. Again, dependingupon the order of addition of reactants to the liquid phase, testcompounds that inhibit complex or that disrupt preformed complexes canbe identified.

In an alternate embodiment of the invention, a homogeneous assay can beused. For example, a preformed complex of the target gene product andthe interactive cellular or extracellular binding partner product isprepared in that either the target gene products or their bindingpartners are labeled, but the signal generated by the label is quencheddue to complex formation (see, e.g., U.S. Pat. No. 4,109,496 thatutilizes this approach for immunoassays). The addition of a testsubstance that competes with and displaces one of the species from thepreformed complex will result in the generation of a signal abovebackground. In this way, test substances that disrupt target geneproduct-binding partner interaction can be identified.

In yet another aspect, the DGAT2 family member proteins can be used as“bait proteins” in a two-hybrid assay or three-hybrid assay (see, e.g.,U.S. Pat. No. 5,283,317; Zervos et al., (1993) Cell 72:223-232; Maduraet al., (1993) J. Biol. Chem. 268:12046-12054; Bartel et al., (1993)Biotechniques 14:920-924; Iwabuchi et al., (1993) Oncogene 8:1693-1696;and Brent WO94/10300), to identify other proteins, which bind to orinteract with DGAT2 family member (“DGAT2 family member-bindingproteins” or “DGAT2 family member-bp”) and are involved in DGAT2 familymember activity. Such DGAT2 family member-bps can be activators orinhibitors of signals by the DGAT2 family member proteins or DGAT2family member targets as, for example, downstream elements of a DGAT2family member-mediated signaling pathway.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a DGAT2 familymember protein is fused to a gene encoding the DNA binding domain of aknown transcription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. (Alternatively the:DGAT2 family member protein can be the fused to the activator domain.)If the “bait” and the “prey” proteins are able to interact, in vivo,forming a DGAT2 family member-dependent complex, the DNA-binding andactivation domains of the transcription factor are brought into closeproximity. This proximity allows transcription of a reporter gene (e.g.,LacZ) which is operably linked to a transcriptional regulatory siteresponsive to the transcription factor. Expression of the reporter genecan be detected and cell colonies containing the functionaltranscription factor can be isolated and used to obtain the cloned genewhich encodes the protein which interacts with the DGAT2 family memberprotein.

In another embodiment, modulators of DGAT2 family member expression areidentified. For example, a cell or cell free mixture is contacted with acandidate compound and the expression of DGAT2 family member mRNA orprotein evaluated relative to the level of expression of DGAT2 familymember mRNA or protein in the absence of the candidate compound. Whenexpression of DGAT2 family member mRNA or protein is greater in thepresence of the candidate compound than in its absence, the candidatecompound is identified as a stimulator of DGAT2 family member mRNA orprotein expression. Alternatively, when expression of DGAT2 familymember mRNA or protein is less (statistically significantly less) in thepresence of the candidate compound than in its absence, the candidatecompound is identified as an inhibitor of DGAT2 family member mRNA orprotein expression. The level of DGAT2 family member mRNA or proteinexpression can be determined by methods described herein for detectingDGAT2 family member mRNA or protein.

In another aspect, the invention pertains to a combination of two ormore of the assays described herein. For example, a modulating agent canbe identified using a cell-based or a cell free assay, and the abilityof the agent to modulate the activity of a DGAT2 family member proteincan be confirmed in vivo, e.g., in an animal.

In another aspect, the methods may be combined and/or a single methodmay be used comparatively with various DGAT2 family members of theinvention in order to identify selective inhibitors of one or more DGAT2family members of the invention. Utilization of suchcombination/comparative assays will allow for the identification ofselective inhibition of particular DGAT2 family member function whichmay be uniquely affected in one or more tissues and or disease states.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein(e.g., a DGAT2 family member modulating agent, an antisense DGAT2 familymember nucleic acid molecule, a DGAT2 family member-specific antibody,or a DGAT2 family member-binding partner) in an appropriate animal modelto determine the efficacy, toxicity, side effects, or mechanism ofaction, of treatment with such an agent. Furthermore, novel agentsidentified by the above-described screening assays can be used fortreatments as described herein.

Detection Assays

Portions or fragments of the nucleic acid sequences identified hereincan be used as polynucleotide reagents. For example, these sequences canbe used to: (i) map their respective genes on a chromosome e.g., tolocate gene regions associated with genetic disease or to associate oneor more DGAT2 family member family members with a disease; (ii) identifyan individual from a minute biological sample (tissue typing); and (iii)aid in forensic identification of a biological sample. Theseapplications are described in the subsections below.

Chromosome Mapping

The DGAT2 family member nucleotide sequences or portions thereof can beused to map the location of the DGAT2 family member genes on achromosome. This process is called chromosome mapping. Chromosomemapping is useful in correlating the DGAT2 family member sequences withgenes associated with disease.

Briefly, DGAT2 family member genes can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp in length) from the DGAT2family member nucleotide sequences. These primers can then be used forPCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human gene correspondingto the DGAT2 family member sequences will yield an amplified fragment.

A panel of somatic cell hybrids in which each cell line contains eithera single human chromosome or a small number of human chromosomes, and afull set of mouse chromosomes, can allow easy mapping of individualgenes to specific human chromosomes. (D'Eustachio P. et al., (1983)Science 220:919-924).

Other mapping strategies e.g., in situ hybridization (described in Fan,Y. et al., (1990) Proc. Natl. Acad. Sci. USA, 87:6223-27), pre-screeningwith labeled flow-sorted chromosomes, and pre-selection by hybridizationto chromosome specific cDNA libraries can be used to map DGAT2 familymember to a chromosomal location.

Fluorescence in situ hybridization (FISH) of a DNA sequence to ametaphase chromosomal spread can further be used to provide a precisechromosomal location in one step. The FISH technique can be used with aDNA sequence as short as 500 or 600 bases. However, clones larger than1,000 bases have a higher likelihood of binding to a unique chromosomallocation with sufficient signal intensity for simple detection.Preferably 1,000 bases, and more preferably 2,000 bases will suffice toget good results at a reasonable amount of time. For a review of thistechnique, see Verma et al., Human Chromosomes: A Manual of BasicTechniques (Pergamon Press, New York 1988).

Reagents for chromosome mapping can be used individually to mark asingle chromosome or a single site on that chromosome, or panels ofreagents can be used for marking multiple sites and/or multiplechromosomes. Reagents corresponding to noncoding regions of the genesactually are preferred for mapping purposes. Coding sequences are morelikely to be conserved within gene families, thus increasing the chanceof cross hybridizations during chromosomal mapping.

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. (Such data are found, for example, in V.McKusick, Mendelian Inheritance in Man, available on-line through JohnsHopkins University Welch Medical Library). The relationship between agene and a disease, mapped to the same chromosomal region, can then beidentified through linkage analysis (co-inheritance of physicallyadjacent genes), described in, for example, Egeland, J. et al., (1987)Nature, 325:783-787.

Moreover, differences in the DNA sequences between individuals affectedand unaffected with a disease associated with the DGAT2 family membergene, can be determined. If a mutation is observed in some or all of theaffected individuals but not in any unaffected individuals, then themutation is likely to be the causative agent of the particular disease.Comparison of affected and unaffected individuals generally involvesfirst looking for structural alterations in the chromosomes, such asdeletions or translocations that are visible from chromosome spreads ordetectable using PCR based on that DNA sequence. Ultimately, completesequencing of genes from several individuals can be performed to confirmthe presence of a mutation and to distinguish mutations frompolymorphisms.

Tissue Typing

DGAT2 family member sequences can be used to identify individuals frombiological samples using, e.g., restriction fragment length polymorphism(RFLP). In this technique, an individual's genomic DNA is digested withone or more restriction enzymes, the fragments separated, e.g., in aSouthern blot, and probed to yield bands for identification. Thesequences of the present invention are useful as additional DNA markersfor RFLP (described in U.S. Pat. No. 5,272,057).

Furthermore, the sequences of the present invention can also be used todetermine the actual base-by-base DNA sequence of selected portions ofan individual's genome. Thus, the DGAT2 family member nucleotidesequences described herein can be used to prepare two PCR primers fromthe 5′ and 3′ ends of the sequences. These primers can then be used toamplify an individual's DNA and subsequently sequence it. Panels ofcorresponding DNA sequences from individuals, prepared in this manner,can provide unique individual identifications, as each individual willhave a unique set of such DNA sequences due to allelic differences.

Allelic variation occurs to some degree in the coding regions of thesesequences, and to a greater degree in the noncoding regions. Each of thesequences described herein can, to some degree, be used as a standardagainst which DNA from an individual can be compared for identificationpurposes. Because greater numbers of polymorphisms occur in thenoncoding regions, fewer sequences are necessary to differentiateindividuals. The noncoding sequences of SEQ ID NO:7 or SEQ ID NO:19 orSEQ ID NO:61 can provide positive individual identification with a panelof perhaps 10 to 1,000 primers which each yield a noncoding amplifiedsequence of 100 bases. If predicted coding sequences, such as those inSEQ ID NO:7 or SEQ ID NO:19 or SEQ ID NO:61 are used, a more appropriatenumber of primers for positive individual identification would be500-2,000.

If a panel of reagents from DGAT2 family member nucleotide sequencesdescribed herein is used to generate a unique identification databasefor an individual, those same reagents can later be used to identifytissue from that individual. Using the unique identification database,positive identification of the individual, living or dead, can be madefrom extremely small tissue samples.

Use of Partial DGAT2 Family Member Sequences in Forensic Biology

DNA-based identification techniques can also be used in forensicbiology. To make such an identification, PCR technology can be used toamplify DNA sequences taken from very small biological samples such astissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, orsemen found at a crime scene. The amplified sequence can then becompared to a standard, thereby allowing identification of the origin ofthe biological sample.

The sequences of the present invention can be used to providepolynucleotide reagents, e.g., PCR primers, targeted to specific loci inthe human genome, which can enhance the reliability of DNA-basedforensic identifications by, for example, providing another“identification marker” (i.e. another DNA sequence that is unique to aparticular individual). As mentioned above, actual base sequenceinformation can be used for identification as an accurate alternative topatterns formed by restriction enzyme generated fragments. Sequencestargeted to noncoding regions of DGAT2 family member sequence (e.g., SEQID NO:7, SEQ ID NO:19, or SEQ ID NO:61 (e.g., fragments derived from thenoncoding regions of SEQ ID NO:7, SEQ ID NO:19 or SEQ ID NO:61 having alength of at least 20 bases, preferably at least 30 bases)) areparticularly appropriate for this use.

The DGAT2 family member nucleotide sequences described herein canfurther be used to provide polynucleotide reagents, e.g., labeled orlabelable probes which can be used in, for example, an in situhybridization technique, to identify a specific tissue, e.g., a tissuecontaining one or more DGAT2 family member activities. This can be veryuseful in cases where a forensic pathologist is presented with a tissueof unknown origin. Panels of such DGAT2 family member probes can be usedto identify tissue by species and/or by organ type.

In a similar fashion, these reagents, e.g., DGAT2 family member primersor probes can be used to screen tissue culture for contamination (i.e.screen for the presence of a mixture of different types of cells in aculture).

Predictive Medicine

The present invention also pertains to the field of predictive medicinein which diagnostic assays, prognostic assays, and monitoring clinicaltrials are used for prognostic (predictive) purposes to thereby treat anindividual.

Generally, the invention provides, a method of determining if a subjectis at risk for a disorder related to a lesion in or the misexpression ofone or more genes which encode a DGAT2 family member.

Such disorders include, e.g., a disorder associated with themisexpression of DGAT2 family member, or lipid metabolism relateddisorder.

The method includes one or more of the following:

detecting, in a tissue of the subject, the presence or absence of amutation which affects the expression of one or more DGAT2 family membergenes, or detecting the presence or absence of a mutation in a regionwhich controls the expression of the gene, e.g., a mutation in the 5′control region;detecting, in a tissue of the subject, the presence or absence of amutation which alters the structure of one or more DGAT2 family membergenes;detecting, in a tissue of the subject, the misexpression of one or moreDGAT2 family member genes, at the mRNA level, e.g., detecting a non-wildtype level of a mRNA;detecting, in a tissue of the subject, the misexpression of the gene, atthe protein level, e.g., detecting a non-wild type level of a DGAT2family member polypeptide.

In preferred embodiments the method includes: ascertaining the existenceof at least one of: a deletion of one or more nucleotides from a DGAT2family member gene; an insertion of one or more nucleotides into a DGAT2family member gene, a point mutation, e.g., a substitution of one ormore nucleotides of the gene, a gross chromosomal rearrangement of thegene, e.g., a translocation, inversion, or deletion.

For example, detecting the genetic lesion can include: (i) providing aprobe/primer including an oligonucleotide containing a region ofnucleotide sequence which hybridizes to a sense or antisense sequencefrom SEQ ID NO:7, SEQ ID NO:19 or SEQ ID NO:61 or naturally occurringmutants thereof or 5′ or 3′ flanking sequences naturally associated witha DGAT2 family member gene; (ii) exposing the probe/primer to nucleicacid of the tissue; and detecting, by hybridization, e.g., in situhybridization, of the probe/primer to the nucleic acid, the presence orabsence of the genetic lesion.

In preferred embodiments detecting the misexpression includesascertaining the existence of at least one of: an alteration in thelevel of a messenger RNA transcript of a DGAT2 family member gene; thepresence of a non-wild type splicing pattern of a messenger RNAtranscript of the gene; or a non-wild type level of DGAT2 family memberexpression.

Methods of the invention can be used prenatally or to determine if asubject's offspring will be at risk for a disorder.

In preferred embodiments the method includes determining the structureof a DGAT2 family member gene, an abnormal structure being indicative ofrisk for the disorder.

In preferred embodiments the method includes contacting a sample fromthe subject with an antibody to a DGAT2 family member protein or anucleic acid, which hybridizes specifically with a DGAT2 family membergene. These and other embodiments are discussed below.

Diagnostic and Prognostic Assays

The presence, level, or absence of a DGAT2 family member protein ornucleic acid in a biological sample can be evaluated by obtaining abiological sample from a test subject and contacting the biologicalsample with a compound or an agent capable of detecting a DGAT2 familymember protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes aDGAT2 family member protein such that the presence of a DGAT2 familymember protein or nucleic acid is detected in the biological sample. Theterm “biological sample” includes tissues, cells and biological fluidsisolated from a subject, as well as tissues, cells and fluids presentwithin a subject. A preferred biological sample is serum.

The level of expression of the DGAT2 family member gene can be measuredin a number of ways, including, but not limited to: measuring the mRNAencoded by the DGAT2 family member genes; measuring the amount ofprotein encoded by the DGAT2 family member genes; or measuring theactivity of the protein encoded by the DGAT2 family member genes.

The level of mRNA corresponding to the DGAT2 family member gene in acell can be determined both by in situ and by in vitro formats.

The isolated mRNA can be used in hybridization or amplification assaysthat include, but are not limited to, Southern or Northern analyses,polymerase chain reaction analyses and probe arrays. One preferreddiagnostic method for the detection of mRNA levels involves contactingthe isolated mRNA with a nucleic acid molecule (probe) that canhybridize to the mRNA encoded by the gene being detected. The nucleicacid probe can be, for example, a full-length DGAT2 family membernucleic acid, such as the nucleic acid of SEQ ID NO:7, SEQ ID NO:19, orSEQ ID NO:61 or a portion thereof, such as an oligonucleotide of atleast 7, 15, 30, 50, 100, 250 or 500 nucleotides in length andsufficient to specifically hybridize under stringent conditions to DGAT2family member mRNA or genomic DNA. Other suitable probes for use in thediagnostic assays are described herein.

In one format, mRNA (or cDNA) is immobilized on a surface and contactedwith the probes, for example by running the isolated mRNA on an agarosegel and transferring the mRNA from the gel to a membrane, such asnitrocellulose. In an alternative format, the probes are immobilized ona surface and the mRNA (or cDNA) is contacted with the probes, forexample, in a two-dimensional gene chip array. A skilled artisan canadapt known mRNA detection methods for use in detecting the level ofmRNA encoded by the DGAT2 family member genes.

The level of mRNA in a sample that is encoded by one of DGAT2 familymember can be evaluated with nucleic acid amplification, e.g., by rtPCR(Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany,1991, Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al., 1989,Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal., 1988, Bio/Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques known in the art. As used herein, amplification primers aredefined as being a pair of nucleic acid molecules that can anneal to 5′or 3′ regions of a gene (plus and minus strands, respectively, orvice-versa) and contain a short region in between. In general,amplification primers are from about 10 to 30 nucleotides in length andflank a region from about 50 to 200 nucleotides in length. Underappropriate conditions and with appropriate reagents, such primerspermit the amplification of a nucleic acid molecule comprising thenucleotide sequence flanked by the primers.

For in situ methods, a cell or tissue sample can be prepared/processedand immobilized on a support, typically a glass slide, and thencontacted with a probe that can hybridize to mRNA that encodes the DGAT2family member gene being analyzed.

In another embodiment, the methods further contacting a control samplewith a compound or agent capable of detecting DGAT2 family member mRNA,or genomic DNA, and comparing the presence of DGAT2 family member mRNAor genomic DNA in the control sample with the presence of DGAT2 familymember mRNA or genomic DNA in the test sample.

A variety of methods can be used to determine the level of proteinencoded by DGAT2 family member. In general, these methods includecontacting an agent that selectively binds to the protein, such as anantibody with a sample, to evaluate the level of protein in the sample.In a preferred embodiment, the antibody bears a detectable label.Antibodies can be polyclonal, or more preferably, monoclonal. An intactantibody, or a fragment thereof (e.g., Fab or F(ab′)₂) can be used. Theterm “labeled”, with regard to the probe or antibody, is intended toencompass direct labeling of the probe or antibody by coupling (i.e.,physically linking) a detectable substance to the probe or antibody, aswell as indirect labeling of the probe or antibody by reactivity with adetectable substance. Examples of detectable substances are providedherein.

The detection methods can be used to detect DGAT2 family member proteinin a biological sample in vitro as well as in vivo. In vitro techniquesfor detection of DGAT2 family member protein include enzyme linkedimmunosorbent assays (ELISAs), immunoprecipitations, immunofluorescence,enzyme immunoassay (EIA), radioimmunoassay (RIA), and Western blotanalysis. In vivo techniques for detection of DGAT2 family memberprotein include introducing into a subject a labeled anti-DGAT2 familymember antibody. For example, the antibody can be labeled with aradioactive marker whose presence and location in a subject can bedetected by standard imaging techniques.

In another embodiment, the methods further include contacting thecontrol sample with a compound or agent capable of detecting DGAT2family member protein, and comparing the presence of DGAT2 family memberprotein in the control sample with the presence of DGAT2 family memberprotein in the test sample.

The invention also includes kits for detecting the presence of DGAT2family member in a biological sample. For example, the kit can include acompound or agent capable of detecting DGAT2 family member protein ormRNA in a biological sample; and a standard. The compound or agent canbe packaged in a suitable container. The kit can further compriseinstructions for using the kit to detect DGAT2 family member protein ornucleic acid.

For antibody-based kits, the kit can include: (1) a first antibody(e.g., attached to a solid support) which binds to a polypeptidecorresponding to a marker of the invention; and, optionally, (2) asecond, different antibody which binds to either the polypeptide or thefirst antibody and is conjugated to a detectable agent.

For oligonucleotide-based kits, the kit can include: (1) anoligonucleotide, e.g., a detectably labeled oligonucleotide, whichhybridizes to a nucleic acid sequence encoding a polypeptidecorresponding to a marker of the invention or (2) a pair of primersuseful for amplifying a nucleic acid molecule corresponding to a markerof the invention. The kit can also includes a buffering agent, apreservative, or a protein-stabilizing agent. The kit can also includescomponents necessary for detecting the detectable agent (e.g., an enzymeor a substrate). The kit can also contain a control sample or a seriesof control samples which can be assayed and compared to the test samplecontained. Each component of the kit can be enclosed within anindividual container and all of the various containers can be within asingle package, along with instructions for interpreting the results ofthe assays performed using the kit.

The diagnostic methods described herein can identify subjects having, orat risk of developing, a disease or disorder associated withmisexpressed or aberrant or unwanted DGAT2 family member expression oractivity. As used herein, the term “unwanted” includes an unwantedphenomenon involved in a biological response such as pain or deregulatedcell proliferation.

In one embodiment, a disease or disorder associated with aberrant orunwanted DGAT2 family member expression or activity is identified. Atest sample is obtained from a subject and DGAT2 family member proteinor nucleic acid (e.g., mRNA or genomic DNA) is evaluated, wherein thelevel, e.g., the presence or absence, of DGAT2 family member protein ornucleic acid is diagnostic for a subject having or at risk of developinga disease or disorder associated with aberrant or unwanted DGAT2 familymember expression or activity. As used herein, a “test sample” refers toa biological sample obtained from a subject of interest, including abiological fluid (e.g., serum), cell sample, or tissue.

The prognostic assays described herein can be used to determine whethera subject can be administered an agent (e.g., an agonist, antagonist,peptidomimetic, protein, peptide, nucleic acid, small molecule, or otherdrug candidate) to treat a disease or disorder associated with aberrantor unwanted DGAT2 family member expression or activity. For example,such methods can be used to determine whether a subject can beeffectively treated with an agent for a cellular growth relateddisorder.

The methods of the invention can also be used to detect geneticalterations in a DGAT2 family member gene, thereby determining if asubject with the altered gene is at risk for a disorder characterized bymisregulation in DGAT2 family member protein activity or nucleic acidexpression, such as a cellular growth related disorder. In preferredembodiments, the methods include detecting, in a sample from thesubject, the presence or absence of a genetic alteration characterizedby at least one of an alteration affecting the integrity of a geneencoding a DGAT2 family member-protein, or the mis-expression of theDGAT2 family member gene. For example, such genetic alterations can bedetected by ascertaining the existence of at least one of 1) a deletionof one or more nucleotides from a DGAT2 family member gene; 2) anaddition of one or more nucleotides to a DGAT2 family member gene; 3) asubstitution of one or more nucleotides of a DGAT2 family member gene,4) a chromosomal rearrangement of a DGAT2 family member gene; 5) analteration in the level of a messenger RNA transcript of a DGAT2 familymember gene, 6) aberrant modification of a DGAT2 family member gene,such as of the methylation pattern of the genomic DNA, 7) the presenceof a non-wild type splicing pattern of a messenger RNA transcript of aDGAT2 family member gene, 8) a non-wild type level of a DGAT2 familymember-protein, 9) allelic loss of a DGAT2 family member gene, and 10)inappropriate post-translational modification of a DGAT2 familymember-protein.

An alteration can be detected without a probe/primer in a polymerasechain reaction, such as anchor PCR or RACE PCR, or, alternatively, in aligation chain reaction (LCR), the latter of which can be particularlyuseful for detecting point mutations in the DGAT2 family member-gene.This method can include the steps of collecting a sample of cells from asubject, isolating nucleic acid (e.g., genomic, mRNA or both) from thesample, contacting the nucleic acid sample with one or more primerswhich specifically hybridize to a DGAT2 family member gene underconditions such that hybridization and amplification of the DGAT2 familymember-gene (if present) occurs, and detecting the presence or absenceof an amplification product, or detecting the size of the amplificationproduct and comparing the length to a control sample. It is anticipatedthat PCR and/or LCR may be desirable to use as a preliminaryamplification step in conjunction with any of the techniques used fordetecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,(1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al., (1988) Bio-Technology 6:1197), or other nucleicacid amplification methods, followed by the detection of the amplifiedmolecules using techniques known to those of skill in the art.

In another embodiment, mutations in a DGAT2 family member gene from asample cell can be identified by detecting alterations in restrictionenzyme cleavage patterns. For example, sample and control DNA isisolated, amplified (optionally), digested with one or more restrictionendonucleases, and fragment length sizes are determined, e.g., by gelelectrophoresis and compared. Differences in fragment length sizesbetween sample and control DNA indicates mutations in the sample DNA.Moreover, the use of sequence specific ribozymes (see, for example, U.S.Pat. No. 5,498,531) can be used to score for the presence of specificmutations by development or loss of a ribozyme cleavage site.

In other embodiments, genetic mutations in DGAT2 family member can beidentified by hybridizing a sample and control nucleic acids, e.g., DNAor RNA, two-dimensional arrays, e.g., chip based arrays. Such arraysinclude a plurality of addresses, each of which is positionallydistinguishable from the other. A different probe is located at eachaddress of the plurality. The arrays can have a high density ofaddresses, e.g., can contain hundreds or thousands of oligonucleotidesprobes (Cronin, M. T. et al., (1996) Human Mutation 7: 244-255; Kozal,M. J. et al., (1996) Nature Medicine 2:753-759). For example, geneticmutations in DGAT2 family member can be identified in two dimensionalarrays containing light-generated DNA probes as described in Cronin, M.T. et al., supra. Briefly, a first hybridization array of probes can beused to scan through long stretches of DNA in a sample and control toidentify base changes between the sequences by making linear arrays ofsequential overlapping probes. This step allows the identification ofpoint mutations. This step is followed by a second hybridization arraythat allows the characterization of specific mutations by using smaller,specialized probe arrays complementary to all variants or mutationsdetected. Each mutation array is composed of parallel probe sets, onecomplementary to the wild-type gene and the other complementary to themutant gene.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence the DGAT2 familymember gene and detect mutations by comparing the sequence of the sampleDGAT2 family member with the corresponding wild-type (control) sequence.Automated sequencing procedures can be utilized when performing thediagnostic assays ((1995) Biotechniques 19:448), including sequencing bymass spectrometry.

Other methods for detecting mutations in the DGAT2 family member geneinclude methods in which protection from cleavage agents is used todetect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers etal., (1985) Science 230:1242; Cotton et al., (1988) Proc. Natl. Acad.Sci. USA 85:4397; Saleeba et al., (1992) Methods Enzymol. 217:286-295).

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in DGAT2 family member cDNAsobtained from samples of cells. For example, the mutY enzyme of E. colicleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLacells cleaves T at G/T mismatches (Hsu et al., (1994) Carcinogenesis15:1657-1662; U.S. Pat. No. 5,459,039).

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations in DGAT2 family member genes. For example,single strand conformation polymorphism (SSCP) may be used to detectdifferences in electrophoretic mobility between mutant and wild typenucleic acids (Orita et al., (1989) Proc. Natl. Acad. Sci. USA: 86:2766,see also Cotton, (1993) Mutat. Res. 285:125-144; and Hayashi, (1992)Genet. Anal. Tech. Appl. 9:73-79). Single-stranded DNA fragments ofsample and control DGAT2 family member nucleic acids will be denaturedand allowed to renature. The secondary structure of single-strandednucleic acids varies according to sequence, the resulting alteration inelectrophoretic mobility enables the detection of even a single basechange. The DNA fragments may be labeled or detected with labeledprobes. The sensitivity of the assay may be enhanced by using RNA(rather than DNA), in which the secondary structure is more sensitive toa change in sequence. In a preferred embodiment, the subject methodutilizes heteroduplex analysis to separate double stranded heteroduplexmolecules on the basis of changes in electrophoretic mobility (Keen etal., (1991) Trends Genet. 7:5).

In yet another embodiment, the movement of mutant or wild-type fragmentsin polyacrylamide gels containing a gradient of denaturant is assayedusing denaturing gradient gel electrophoresis (DGGE) (Myers et al.,(1985) Nature 313:495). When DGGE is used as the method of analysis, DNAwill be modified to insure that it does not completely denature, forexample by adding a GC clamp of approximately 40 bp of high-meltingGC-rich DNA by PCR. In a further embodiment, a temperature gradient isused in place of a denaturing gradient to identify differences in themobility of control and sample DNA (Rosenbaum and Reissner, (1987)Biophys. Chem. 265:12753).

Examples of other techniques for detecting point mutations include, butare not limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension (Saiki et al., (1986)Nature 324:163); Saiki et al., (1989) Proc. Natl. Acad. Sci. USA86:6230).

Alternatively, allele specific amplification technology which depends onselective PCR amplification may be used in conjunction with the instantinvention. Oligonucleotides used as primers for specific amplificationmay carry the mutation of interest in the center of the molecule (sothat amplification depends on differential hybridization) (Gibbs et al.,(1989) Nucleic Acids Res. 17:2437-2448) or at the extreme 3′ end of oneprimer where, under appropriate conditions, mismatch can prevent, orreduce polymerase extension (Prossner, (1993) Tibtech 11:238). Inaddition it may be desirable to introduce a novel restriction site inthe region of the mutation to create cleavage-based detection (Gaspariniet al., (1992) Mol. Cell. Probes 6:1). It is anticipated that in certainembodiments amplification may also be performed using Taq ligase foramplification (Barany, (1991) Proc. Natl. Acad. Sci. USA 88:189). Insuch cases, ligation will occur only if there is a perfect match at the3′ end of the 5′ sequence making it possible to detect the presence of aknown mutation at a specific site by looking for the presence or absenceof amplification.

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acidor antibody reagent described herein, which may be conveniently used,e.g., in clinical settings to diagnose patients exhibiting symptoms orfamily history of a disease or illness involving a DGAT2 family membergene.

Use of DGAT2 Family Member Molecules as Surrogate Markers

The DGAT2 family member molecules of the invention are also useful asmarkers of disorders or disease states, as markers for precursors ofdisease states, as markers for predisposition of disease states, asmarkers of drug activity, or as markers of the pharmacogenomic profileof a subject. Using the methods described herein, the presence, absenceand/or quantity of the DGAT2 family member molecules of the inventionmay be detected, and may be correlated with one or more biologicalstates in vivo. For example, the DGAT2 family member molecules of theinvention may serve as surrogate markers for one or more disorders ordisease states or for conditions leading up to disease states. As usedherein, a “surrogate marker” is an objective biochemical marker whichcorrelates with the absence or presence of a disease or disorder, orwith the progression of a disease or disorder (e.g., with the presenceor absence of a tumor). The presence or quantity of such markers isindependent of the disease. Therefore, these markers may serve toindicate whether a particular course of treatment is effective inlessening a disease state or disorder. Surrogate markers are ofparticular use when the presence or extent of a disease state ordisorder is difficult to assess through standard methodologies (e.g.,early stage tumors), or when an assessment of disease progression isdesired before a potentially dangerous clinical endpoint is reached(e.g., an assessment of cardiovascular disease may be made usingcholesterol levels as a surrogate marker, and an analysis of HIVinfection may be made using HIV RNA levels as a surrogate marker, wellin advance of the undesirable clinical outcomes of myocardial infarctionor fully-developed AIDS). Examples of the use of surrogate markers inthe art include: Koomen et al. (2000) J. Mass. Spectrom. 35: 258-264;and James (1994) AIDS Treatment News Archive 209.

The DGAT2 family member molecules of the invention are also useful aspharmacodynamic markers. As used herein, a “pharmacodynamic marker” isan objective biochemical marker which correlates specifically with drugeffects. The presence or quantity of a pharmacodynamic marker is notrelated to the disease state or disorder for which the drug is beingadministered; therefore, the presence or quantity of the marker isindicative of the presence or activity of the drug in a subject. Forexample, a pharmacodynamic marker may be indicative of the concentrationof the drug in a biological tissue, in that the marker is eitherexpressed or transcribed or not expressed or transcribed in that tissuein relationship to the level of the drug. In this fashion, thedistribution or uptake of the drug may be monitored by thepharmacodynamic marker. Similarly, the presence or quantity of thepharmacodynamic marker may be related to the presence or quantity of themetabolic product of a drug, such that the presence or quantity of themarker is indicative of the relative breakdown rate of the drug in vivo.Pharmacodynamic markers are of particular use in increasing thesensitivity of detection of drug effects, particularly when the drug isadministered in low doses. Since even a small amount of a drug may besufficient to activate multiple rounds of marker (e.g., a DGAT2 familymember marker) transcription or expression, the amplified marker may bein a quantity which is more readily detectable than the drug itself.Also, the marker may be more easily detected due to the nature of themarker itself; for example, using the methods described herein,anti-DGAT2 family member antibodies may be employed in an immune-baseddetection system for a DGAT2 family member protein marker, or DGAT2family member-specific radiolabeled probes may be used to detect a DGAT2family member mRNA marker. Furthermore, the use of a pharmacodynamicmarker may offer mechanism-based prediction of risk due to drugtreatment beyond the range of possible direct observations. Examples ofthe use of pharmacodynamic markers in the art include: Matsuda et al.U.S. Pat. No. 6,033,862; Hattis et al. (1991) Env. Health Perspect. 90:229-238; Schentag (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3:S21-S24; and Nicolau (1999) Am. J. Health-Syst. Pharm. 56 Suppl. 3:S16-S20.

The DGAT2 family member molecules of the invention are also useful aspharmacogenomic markers. As used herein, a “pharmacogenomic marker” isan objective biochemical marker which correlates with a specificclinical drug response or susceptibility in a subject (see, e.g., McLeodet al. (1999) Eur. J. Cancer 35(12): 1650-1652). The presence orquantity of the pharmacogenomic marker is related to the predictedresponse of the subject to a specific drug or class of drugs prior toadministration of the drug. By assessing the presence or quantity of oneor more pharmacogenomic markers in a subject, a drug therapy which ismost appropriate for the subject, or which is predicted to have agreater degree of success, may be selected. For example, based on thepresence or quantity of RNA, or protein (e.g., DGAT2 family memberprotein or RNA) for specific tumor markers in a subject, a drug orcourse of treatment may be selected that is optimized for the treatmentof the specific tumor likely to be present in the subject. Similarly,the presence or absence of a specific sequence mutation in DGAT2 familymember DNA may correlate DGAT2 family member drug response. The use ofpharmacogenomic markers therefore permits the application of the mostappropriate treatment for each subject without having to administer thetherapy.

Pharmaceutical Compositions

The nucleic acid and polypeptides, fragments thereof, as well asanti-DGAT2 family member antibodies (also referred to herein as “activecompounds”) of the invention can be incorporated into pharmaceuticalcompositions. Such compositions typically include the nucleic acidmolecule, protein, or antibody and a pharmaceutically acceptablecarrier. As used herein the language “pharmaceutically acceptablecarrier” includes solvents, dispersion media, coatings, antibacterialand antifungal agents, isotonic and absorption delaying agents, and thelike, compatible with pharmaceutical administration. Supplementaryactive compounds can also be incorporated into the compositions.

A pharmaceutical composition is formulated to be compatible with itsintended route of administration. Examples of routes of administrationinclude parenteral, e.g., intravenous, intradermal, subcutaneous, oral(e.g., inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycolor other synthetic solvents; antibacterial agents such as benzyl alcoholor methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents for theadjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringability exists. It should be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyetheylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol, sorbitol, sodium chloride in thecomposition. Prolonged absorption of the injectable compositions can bebrought about by including in the composition an agent which delaysabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle which containsa basic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and freeze-drying which yields a powder of the activeingredient plus any additional desired ingredient from a previouslysterile-filtered solution thereof.

Oral compositions generally include an inert diluent or an ediblecarrier. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules, e.g., gelatin capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash.Pharmaceutically compatible binding agents, and/or adjuvant materialscan be included as part of the composition. The tablets, pills,capsules, troches and the like can contain any of the followingingredients, or compounds of a similar nature: a binder such asmicrocrystalline cellulose, gum tragacanth or gelatin; an excipient suchas starch or lactose, a disintegrating agent such as alginic acid,Primogel, or corn starch; a lubricant such as magnesium stearate orSterotes; a glidant such as colloidal silicon dioxide; a sweeteningagent such as sucrose or saccharin; or a flavoring agent such aspeppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The compounds can also be prepared in the form of suppositories (e.g.,with conventional suppository bases such as cocoa butter and otherglycerides) or retention enemas for rectal delivery.

In one embodiment, the active compounds are prepared with carriers thatwill protect the compound against rapid elimination from the body, suchas a controlled release formulation, including implants andmicroencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is advantageous to formulate oral or parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subject to be treated; each unitcontaining a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds which exhibit high therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects may be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose may beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

As defined herein, a therapeutically effective amount of protein orpolypeptide (i.e., an effective dosage) ranges from about 0.001 to 30mg/kg body weight, preferably about 0.01 to 25 mg/kg body weight, morepreferably about 0.1 to 20 mg/kg body weight, and even more preferablyabout 1 to 10 mg/kg, 2 to 9 mg/kg, 3 to 8 mg/kg, 4 to 7 mg/kg, or 5 to 6mg/kg body weight. The protein or polypeptide can be administered onetime per week for between about 1 to 10 weeks, preferably between 2 to 8weeks, more preferably between about 3 to 7 weeks, and even morepreferably for about 4, 5, or 6 weeks. The skilled artisan willappreciate that certain factors may influence the dosage and timingrequired to effectively treat a subject, including but not limited tothe severity of the disease or disorder, previous treatments, thegeneral health and/or age of the subject, and other diseases present.Moreover, treatment of a subject with a therapeutically effective amountof a protein, polypeptide, or antibody can include a single treatmentor, preferably, can include a series of treatments.

For antibodies, the preferred dosage is 0.1 mg/kg of body weight(generally 10 mg/kg to 20 mg/kg). If the antibody is to act in thebrain, a dosage of 50 mg/kg to 100 mg/kg is usually appropriate.Generally, partially human antibodies and fully human antibodies have alonger half-life within the human body than other antibodies.Accordingly, lower dosages and less frequent administration is oftenpossible. Modifications such as lipidation can be used to stabilizeantibodies and to enhance uptake and tissue penetration (e.g., into thebrain). A method for lipidation of antibodies is described by Cruikshanket al., ((1997) J. Acquired Immune Deficiency Syndromes and HumanRetrovirology 14:193).

The present invention encompasses agents which modulate expression oractivity. An agent may, for example, be a small molecule. For example,such small molecules include, but are not limited to, peptides,peptidomimetics (e.g., peptoids), amino acids, amino acid analogs,polynucleotides, polynucleotide analogs, nucleotides, nucleotideanalogs, organic or inorganic compounds (i.e., including heteroorganicand organometallic compounds) having a molecular weight less than about10,000 grams per mole, organic or inorganic compounds having a molecularweight less than about 5,000 grams per mole, organic or inorganiccompounds having a molecular weight less than about 1,000 grams permole, organic or inorganic compounds having a molecular weight less thanabout 500 grams per mole, and salts, esters, and other pharmaceuticallyacceptable forms of such compounds.

Exemplary doses include milligram or microgram amounts of the smallmolecule per kilogram of subject or sample weight (e.g., about 1microgram per kilogram to about 500 milligrams per kilogram, about 100micrograms per kilogram to about 5 milligrams per kilogram, or about 1microgram per kilogram to about 50 micrograms per kilogram. It isfurthermore understood that appropriate doses of a small molecule dependupon the potency of the small molecule with respect to the expression oractivity to be modulated. When one or more of these small molecules isto be administered to an animal (e.g., a human) in order to modulateexpression or activity of a polypeptide or nucleic acid of theinvention, a physician, veterinarian, or researcher may, for example,prescribe a relatively low dose at first, subsequently increasing thedose until an appropriate response is obtained. In addition, it isunderstood that the specific dose level for any particular animalsubject will depend upon a variety of factors including the activity ofthe specific compound employed, the age, body weight, general health,gender, and diet of the subject, the time of administration, the routeof administration, the rate of excretion, any drug combination, and thedegree of expression or activity to be modulated.

An antibody (or fragment thereof) may be conjugated to a therapeuticmoiety such as a cytotoxin, a therapeutic agent or a radioactive metalion. A cytotoxin or cytotoxic agent includes any agent that isdetrimental to cells. Examples include taxol, cytochalasin B, gramicidinD, ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the drug moiety is not to be construed as limitedto classical chemical therapeutic agents. For example, the drug moietymay be a protein or polypeptide possessing a desired biologicalactivity. Such proteins may include, for example, a toxin such as abrin,ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such astumor necrosis factor, alpha.-interferon, .beta.-interferon, nervegrowth factor, platelet derived growth factor, tissue plasminogenactivator; or, biological response modifiers such as, for example,lymphokines, interleukin-1 (“IL-1”), interleukin-2 (“IL-2”),interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor(“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or othergrowth factors.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980.

The nucleic acid molecules of the invention can be inserted into vectorsand used as gene therapy vectors. Gene therapy vectors can be deliveredto a subject by, for example, intravenous injection, localadministration (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (see e.g., Chen et al., (1994) Proc. Natl. Acad. Sci. USA91:3054-3057). The pharmaceutical preparation of the gene therapy vectorcan include the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells which producethe gene delivery system.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

Methods of Treatment:

The present invention provides for both prophylactic and therapeuticmethods of treating a subject at risk of (or susceptible to) a disorderor having a disorder associated with aberrant or unwanted DGAT2 familymember expression or activity. With regards to both prophylactic andtherapeutic methods of treatment, such treatments may be specificallytailored or modified, based on knowledge obtained from the field ofpharmacogenomics. “Pharmacogenomics”, as used herein, refers to theapplication of genomics technologies such as gene sequencing,statistical genetics, and gene expression analysis to drugs in clinicaldevelopment and on the market. More specifically, the term refers thestudy of how a patient's genes determine his or her response to a drug(e.g., a patient's “drug response phenotype”, or “drug responsegenotype”.) Thus, another aspect of the invention provides methods fortailoring an individual's prophylactic or therapeutic treatment witheither the DGAT2 family member molecules of the present invention orDGAT2 family member modulators according to that individual's drugresponse genotype. Pharmacogenomics allows a clinician or physician totarget prophylactic or therapeutic treatments to patients who will mostbenefit from the treatment and to avoid treatment of patients who willexperience toxic drug-related side effects.

In one aspect, the invention provides a method for preventing in asubject, a disease or condition associated with an aberrant or unwantedDGAT2 family member expression or activity, by administering to thesubject a DGAT2 family member or an agent which modulates expression ofDGAT2 family member or at least one DGAT2 family member activity.Subjects at risk for a disease which is caused or contributed to byaberrant or unwanted DGAT2 family member expression or activity can beidentified by, for example, any or a combination of diagnostic orprognostic assays as described herein. Administration of a prophylacticagent can occur prior to the manifestation of symptoms characteristic ofthe DGAT2 family member aberrance, such that a disease or disorder isprevented or, alternatively, delayed in its progression. Depending onthe type of DGAT2 family member aberrance, for example, a DGAT2 familymember, DGAT2 family member agonist or DGAT2 family member antagonistagent can be used for treating the subject. The appropriate agent can bedetermined based on screening assays described herein.

It is possible that some DGAT2 family member disorders can be caused, atleast in part, by an abnormal level of gene product, or by the presenceof a gene product exhibiting abnormal activity. As such, the reductionin the level and/or activity of such gene products would bring about theamelioration of disorder symptoms.

As discussed, successful treatment of DGAT2 family member disorders canbe brought about by techniques that serve to inhibit the expression oractivity of target gene products. For example, compounds, e.g., an agentidentified using one or more assays described above, that proves toexhibit negative modulatory activity, can be used in accordance with theinvention to prevent and/or ameliorate symptoms of DGAT2 family memberrelated disorders (e.g., obesity, diabetes, triglyceride storagedisorders). Such molecules can include, but are not limited to peptides,phosphopeptides, small organic or inorganic molecules, or antibodies(including, for example, polyclonal, monoclonal, humanized,anti-idiotypic, chimeric or single chain antibodies, and FAb, F(ab′)₂and FAb expression library fragments, scFV molecules, andepitope-binding fragments thereof).

Further, antisense and ribozyme molecules that inhibit expression of thetarget gene can also be used in accordance with the invention to reducethe level of target gene expression, thus effectively reducing the levelof target gene activity. Still further, triple helix molecules can beutilized in reducing the level of target gene activity. Antisense,ribozyme and triple helix molecules are discussed above.

It is possible that the use of antisense, ribozyme, and/or triple helixmolecules to reduce or inhibit mutant gene expression can also reduce orinhibit the transcription (triple helix) and/or translation (antisense,ribozyme) of mRNA produced by normal target gene alleles, such that theconcentration of normal target gene product present can be lower than isnecessary for a normal phenotype. In such cases, nucleic acid moleculesthat encode and express target gene polypeptides exhibiting normaltarget gene activity can be introduced into cells via gene therapymethod. Alternatively, in instances in that the target gene encodes anextracellular protein, it can be preferable to co-administer normaltarget gene protein into the cell or tissue in order to maintain therequisite level of cellular or tissue target gene activity.

Another method by which nucleic acid molecules may be utilized intreating or preventing a disease characterized by DGAT2 family memberexpression is through the use of aptamer molecules specific for DGAT2family member protein. Aptamers are nucleic acid molecules having atertiary structure which permits them to specifically bind to proteinligands (see, e.g., Osborne, et al., Curr. Opin. Chem. Biol. 1997, 1(1):5-9; and Patel, D. J., Curr. Opin. Chem. Biol. 1997 Jun.; 1(1):32-46).Since nucleic acid molecules may in many cases be more convenientlyintroduced into target cells than therapeutic protein molecules may be,aptamers offer a method by which DGAT2 family member protein activitymay be specifically decreased without the introduction of drugs or othermolecules which may have pluripotent effects.

Antibodies can be generated that are both specific for target geneproduct and that reduce target gene product activity. Such antibodiesmay, therefore, by administered in instances whereby negative modulatorytechniques are appropriate for the treatment of DGAT2 family memberdisorders. For a description of antibodies, see the Antibody sectionabove.

In circumstances wherein injection of an animal or a human subject witha DGAT2 family member protein or epitope for stimulating antibodyproduction is harmful to the subject, it is possible to generate animmune response against DGAT2 family member through the use ofanti-idiotypic antibodies (see, for example, Herlyn, D., Ann. Med. 1999;31(1):66-78; and Bhattacharya-Chatterjee, M., and Foon, K. A., CancerTreat. Res. 1998; 94:51-68). If an anti-idiotypic antibody is introducedinto a mammal or human subject, it should stimulate the production ofanti-anti-idiotypic antibodies, which should be specific to the DGAT2family member protein. Vaccines directed to a disease characterized byDGAT2 family member expression may also be generated in this fashion.

In instances where the target antigen is intracellular and wholeantibodies are used, internalizing antibodies may be preferred.Lipofectin or liposomes can be used to deliver the antibody or afragment of the Fab region that binds to the target antigen into cells.Where fragments of the antibody are used, the smallest inhibitoryfragment that binds to the target antigen is preferred. For example,peptides having an amino acid sequence corresponding to the Fv region ofthe antibody can be used. Alternatively, single chain neutralizingantibodies that bind to intracellular target antigens can also beadministered. Such single chain antibodies can be administered, forexample, by expressing nucleotide sequences encoding single-chainantibodies within the target cell population (see e.g., Marasco et al.,(1993, Proc. Natl. Acad. Sci. USA 90:7889-7893).

The identified compounds that inhibit target gene expression, synthesisand/or activity can be administered to a patient at therapeuticallyeffective doses to prevent, treat or ameliorate DGAT2 family memberdisorders. A therapeutically effective dose refers to that amount of thecompound sufficient to result in amelioration of symptoms of thedisorders.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit large therapeutic indices are preferred. Whilecompounds that exhibit toxic side effects can be used, care should betaken to design a delivery system that targets such compounds to thesite of affected tissue in order to minimize potential damage touninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED₅₀ with little or no toxicity. Thedosage can vary within this range depending upon the dosage formemployed and the route of administration utilized. For any compound usedin the method of the invention, the therapeutically effective dose canbe estimated initially from cell culture assays. A dose can beformulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound that achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma can bemeasured, for example, by high performance liquid chromatography.

Another example of determination of effective dose for an individual isthe ability to directly assay levels of “free” and “bound” compound inthe serum of the test subject. Such assays may utilize antibody mimicsand/or “biosensors” that have been created through molecular imprintingtechniques. The compound which is able to modulate DGAT2 family memberactivity is used as a template, or “imprinting molecule”, to spatiallyorganize polymerizable monomers prior to their polymerization withcatalytic reagents. The subsequent removal of the imprinted moleculeleaves a polymer matrix which contains a repeated “negative image” ofthe compound and is able to selectively rebind the molecule underbiological assay conditions. A detailed review of this technique can beseen in Ansell, R. J. et al., (1996) Current Opinion in Biotechnology7:89-94 and in Shea, K. J., (1994) Trends in Polymer Science 2:166-173.Such “imprinted” affinity matrixes are amenable to ligand-bindingassays, whereby the immobilized monoclonal antibody component isreplaced by an appropriately imprinted matrix. An example of the use ofsuch matrixes in this way can be seen in Vlatakis, G. et al., (1993)Nature 361:645-647. Through the use of isotope-labeling, the “free”concentration of compound which modulates the expression or activity ofDGAT2 family member can be readily monitored and used in calculations ofIC₅₀.

Such “imprinted” affinity matrixes can also be designed to includefluorescent groups whose photon-emitting properties measurably changeupon local and selective binding of target compound. These changes canbe readily assayed in real time using appropriate fiberoptic devices, inturn allowing the dose in a test subject to be quickly optimized basedon its individual IC₅₀. A rudimentary example of such a “biosensor” isdiscussed in Kriz, D. et al., (1995) Analytical Chemistry 67:2142-2144.

Another aspect of the invention pertains to methods of modulating DGAT2family member expression or activity for therapeutic purposes.Accordingly, in an exemplary embodiment, the modulatory method of theinvention involves contacting a cell with a DGAT2 family member or agentthat modulates one or more of the activities of DGAT2 family memberprotein activity associated with the cell. An agent that modulates DGAT2family member protein activity can be an agent as described herein, suchas a nucleic acid or a protein, a naturally-occurring target molecule ofa DGAT2 family member protein (e.g., a DGAT2 family member substrate orreceptor), a DGAT2 family member antibody, a DGAT2 family member agonistor antagonist, a peptidomimetic of a DGAT2 family member agonist orantagonist, or other small molecule.

In one embodiment, the agent stimulates one or more DGAT2 family memberactivities. Examples of such stimulatory agents include active DGAT2family member protein and a nucleic acid molecule encoding DGAT2 familymember. In another embodiment, the agent inhibits one or more DGAT2family member activities. Examples of such inhibitory agents includeantisense DGAT2 family member nucleic acid molecules, anti-DGAT2 familymember antibodies, and DGAT2 family member inhibitors. These modulatorymethods can be performed in vitro (e.g., by culturing the cell with theagent) or, alternatively, in vivo (e.g., by administering the agent to asubject). As such, the present invention provides methods of treating anindividual afflicted with a disease or disorder characterized byaberrant or unwanted expression or activity of a DGAT2 family memberprotein or nucleic acid molecule. In one embodiment, the method involvesadministering an agent (e.g., an agent identified by a screening assaydescribed herein), or combination of agents that modulates (e.g.,upregulates or downregulates) DGAT2 family member expression oractivity. In another embodiment, the method involves administering aDGAT2 family member protein or nucleic acid molecule as therapy tocompensate for reduced, aberrant, or unwanted DGAT2 family memberexpression or activity.

Stimulation of DGAT2 family member activity is desirable in situationsin which DGAT2 family member is abnormally downregulated and/or in whichincreased DGAT2 family member activity is likely to have a beneficialeffect. For example, stimulation of DGAT2 family member activity isdesirable in situations in which a DGAT2 family member is downregulatedand/or in which increased DGAT2 family member activity is likely to havea beneficial effect. Likewise, inhibition of DGAT2 family memberactivity is desirable in situations in which DGAT2 family member isabnormally upregulated and/or in which decreased DGAT2 family memberactivity is likely to have a beneficial effect.

The DGAT2 family member molecules can act as novel diagnostic targetsand therapeutic agents for controlling one or more of metabolicdisorders, liver disorders, cellular proliferative and/ordifferentiative disorders, cardiovascular disorders, as described above.

Diseases of metabolic imbalance include, but are not limited to,obesity, lipid disorders including hyperlipidemia, and diabetes.

Pharmacogenomics

The DGAT2 family member molecules of the present invention, as well asagents, or modulators which have a stimulatory or inhibitory effect onDGAT2 family member activity (e.g., DGAT2 family member gene expression)as identified by a screening assay described herein can be administeredto individuals to treat (prophylactically or therapeutically) DGAT2family member associated disorders (e.g., cellular growth relateddisorders) associated with aberrant or unwanted DGAT2 family memberactivity. In conjunction with such treatment, pharmacogenomics (i.e.,the study of the relationship between an individual's genotype and thatindividual's response to a foreign compound or drug) may be considered.Differences in metabolism of therapeutics can lead to severe toxicity ortherapeutic failure by altering the relation between dose and bloodconcentration of the pharmacologically active drug. Thus, a physician orclinician may consider applying knowledge obtained in relevantpharmacogenomics studies in determining whether to administer a DGAT2family member molecule or DGAT2 family member modulator as well astailoring the dosage and/or therapeutic regimen of treatment with aDGAT2 family member molecule or DGAT2 family member modulator.

Pharmacogenomics deals with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, for example, Eichelbaum, M. et al.(1996) Clin. Exp. Pharmacol. Physiol. 23(10-11):983-985 and Linder, M.W. et al. (1997) Clin. Chem. 43(2):254-266. In general, two types ofpharmacogenetic conditions can be differentiated. Genetic conditionstransmitted as a single factor altering the way drugs act on the body(altered drug action) or genetic conditions transmitted as singlefactors altering the way the body acts on drugs (altered drugmetabolism). These pharmacogenetic conditions can occur either as raregenetic defects or as naturally-occurring polymorphisms. For example,glucose-6-phosphate dehydrogenase deficiency (G6PD) is a commoninherited enzymopathy in which the main clinical complication ishaemolysis after ingestion of oxidant drugs (anti-malarials,sulfonamides, analgesics, nitrofurans) and consumption of fava beans.

One pharmacogenomics approach to identifying genes that predict drugresponse, known as “a genome-wide association”, relies primarily on ahigh-resolution map of the human genome consisting of already knowngene-related markers (e.g., a “bi-allelic” gene marker map whichconsists of 60,000-100,000 polymorphic or variable sites on the humangenome, each of which has two variants.) Such a high-resolution geneticmap can be compared to a map of the genome of each of a statisticallysignificant number of patients taking part in a Phase II/III drug trialto identify markers associated with a particular observed drug responseor side effect. Alternatively, such a high-resolution map can begenerated from a combination of some ten million known single nucleotidepolymorphisms (SNPs) in the human genome. As used herein, a “SNP” is acommon alteration that occurs in a single nucleotide base in a stretchof DNA. For example, a SNP may occur once per every 1000 bases of DNA. ASNP may be involved in a disease process, however, the vast majority maynot be disease-associated. Given a genetic map based on the occurrenceof such SNPs, individuals can be grouped into genetic categoriesdepending on a particular pattern of SNPs in their individual genome. Insuch a manner, treatment regimens can be tailored to groups ofgenetically similar individuals, taking into account traits that may becommon among such genetically similar individuals.

Alternatively, a method termed the “candidate gene approach”, can beutilized to identify genes that predict drug response. According to thismethod, if a gene that encodes a drug's target is known (e.g., a DGAT2family member protein of the present invention), all common variants ofthat gene can be fairly easily identified in the population and it canbe determined if having one version of the gene versus another isassociated with a particular drug response.

Alternatively, a method termed the “gene expression profiling”, can beutilized to identify genes that predict drug response. For example, thegene expression of an animal dosed with a drug (e.g., a DGAT2 familymember molecule or DGAT2 family member modulator of the presentinvention) can give an indication whether gene pathways related totoxicity have been turned on.

Information generated from more than one of the above pharmacogenomicsapproaches can be used to determine appropriate dosage and treatmentregimens for prophylactic or therapeutic treatment of an individual.This knowledge, when applied to dosing or drug selection, can avoidadverse reactions or therapeutic failure and thus enhance therapeutic orprophylactic efficiency when treating a subject with a DGAT2 familymember molecule or DGAT2 family member modulator, such as a modulatoridentified by one of the exemplary screening assays described herein.

The present invention further provides methods for identifying newagents, or combinations, that are based on identifying agents thatmodulate the activity of one or more of the gene products encoded by oneor more of the DGAT2 family member genes of the present invention,wherein these products may be associated with resistance of the cells toa therapeutic agent. Specifically, the activity of the proteins encodedby the DGAT2 family member genes of the present invention can be used asa basis for identifying agents for overcoming agent resistance. Byblocking the activity of one or more of the resistance proteins, targetcells, e.g., adipocytes, will become sensitive to treatment with anagent that the unmodified target cells were resistant to.

Monitoring the influence of agents (e.g., drugs) on the expression oractivity of a DGAT2 family member protein can be applied in clinicaltrials. For example, the effectiveness of an agent determined by ascreening assay as described herein to increase DGAT2 family member geneexpression, protein levels, or upregulate DGAT2 family member activity,can be monitored in clinical trials of subjects exhibiting decreasedDGAT2 family member gene expression, protein levels, or downregulatedDGAT2 family member activity. Alternatively, the effectiveness of anagent determined by a screening assay to decrease DGAT2 family membergene expression, protein levels, or downregulate DGAT2 family memberactivity, can be monitored in clinical trials of subjects exhibitingincreased DGAT2 family member gene expression, protein levels, orupregulated DGAT2 family member activity. In such clinical trials, theexpression or activity of a DGAT2 family member gene, and preferably,other genes that have been implicated in, for example, a DGAT2 familymember-associated disorder can be used as a “read out” or markers of thephenotype of a particular cell.

Other Embodiments

In another aspect, the invention features, a method of analyzing aplurality of capture probes. The method can be used, e.g., to analyzegene expression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the plurality,and each address of the plurality having a unique capture probe, e.g., anucleic acid or peptide sequence; contacting the array with a DGAT2family member, preferably purified, nucleic acid, preferably purified,polypeptide, preferably purified, or antibody, and thereby evaluatingthe plurality of capture probes. Binding, e.g., in the case of a nucleicacid, hybridization with a capture probe at an address of the plurality,is detected, e.g., by signal generated from a label attached to one ormore DGAT2 family member nucleic acids, polypeptides, or antibodies.

The capture probes can be a set of nucleic acids from a selected sample,e.g., a sample of nucleic acids derived from a control or non-stimulatedtissue or cell.

The method can include contacting the DGAT2 family member nucleic acid,polypeptide, or antibody with a first array having a plurality ofcapture probes and a second array having a different plurality ofcapture probes. The results of each hybridization can be compared, e.g.,to analyze differences in expression between a first and second sample.The first plurality of capture probes can be from a control sample,e.g., a wild type, normal, or non-diseased, non-stimulated, sample,e.g., a biological fluid, tissue, or cell sample. The second pluralityof capture probes can be from an experimental sample, e.g., a mutanttype, at risk, disease-state or disorder-state, or stimulated, sample,e.g., a biological fluid, tissue, or cell sample.

The plurality of capture probes can be a plurality of nucleic acidprobes each of which specifically hybridizes, with an allele of a DGAT2family member molecule. Such methods can be used to diagnose a subject,e.g., to evaluate risk for a disease or disorder, to evaluatesuitability of a selected treatment for a subject, to evaluate whether asubject has a disease or disorder. DGAT2 family member is associatedwith triglyceride biosynthesis or activity, thus it is useful fordisorders associated with abnormal lipid metabolism.

The method can be used to detect SNPs, as described above.

In another aspect, the invention features, a method of analyzing aplurality of probes. The method is useful, e.g., for analyzing geneexpression. The method includes: providing a two dimensional arrayhaving a plurality of addresses, each address of the plurality beingpositionally distinguishable from each other address of the pluralityhaving a unique capture probe, e.g., wherein the capture probes are froma cell or subject which express or mis express one or more DGAT2 familymember molecules of the invention or from a cell or subject in which aDGAT2 family member mediated response has been elicited, e.g., bycontact of the cell with one or more DGAT2 family member nucleic acidsor proteins, or administration to the cell or subject DGAT2 familymember nucleic acids or proteins; contacting the array with one or moreinquiry probe, wherein an inquiry probe can be a nucleic acid,polypeptide, or antibody (which is preferably other than DGAT2 familymember nucleic acid, polypeptide, or antibody); providing a twodimensional array having a plurality of addresses, each address of theplurality being positionally distinguishable from each other address ofthe plurality, and each address of the plurality having a unique captureprobe, e.g., wherein the capture probes are from a cell or subject whichdoes not express DGAT2 family member (or does not express as highly asin the case of the DGAT2 family member positive plurality of captureprobes) or from a cell or subject which in which a DGAT2 family membermediated response has not been elicited (or has been elicited to alesser extent than in the first sample); contacting the array with oneor more inquiry probes (which is preferably other than a DGAT2 familymember nucleic acid, polypeptide, or antibody), and thereby evaluatingthe plurality of capture probes. Binding, e.g., in the case of a nucleicacid, hybridization with a capture probe at an address of the plurality,is detected, e.g., by signal generated from a label attached to thenucleic acid, polypeptide, or antibody.

In another aspect, the invention features, a method of analyzing a DGAT2family member, e.g., analyzing structure, function, or relatedness toother nucleic acid or amino acid sequences. The method includes:providing a DGAT2 family member nucleic acid or amino acid sequence;comparing the DGAT2 family member sequence with one or more preferably aplurality of sequences from a collection of sequences, e.g., a nucleicacid or protein sequence database; to thereby analyze DGAT2 familymember.

Preferred databases include GenBank™. The method can include evaluatingthe sequence identity between a DGAT2 family member sequence and adatabase sequence. The method can be performed by accessing the databaseat a second site, e.g., over the internet.

In another aspect, the invention features, a set of oligonucleotides,useful, e.g., for identifying SNP's, or identifying specific alleles ofDGAT2 family members. The set includes a plurality of oligonucleotides,each of which has a different nucleotide at an interrogation position,e.g., an SNP or the site of a mutation. In a preferred embodiment, theoligonucleotides of the plurality identical in sequence with one another(except for differences in length). The oligonucleotides can be providedwith different labels, such that an oligonucleotides which hybridizes toone allele provides a signal that is distinguishable from anoligonucleotides which hybridizes to a second allele.

This invention is further illustrated by the following examples, whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication are incorporated herein by reference.

EXAMPLES Example 1 Identification and Characterization of Human DGAT2family member cDNAs and Proteins

A number of gene sequences were identified which have homology to theDGAT2 sequences. The human DGAT2, (herein referred to as 86606) sequenceis depicted in SEQ ID NO:9, which is approximately 2428 nucleotides longincluding untranslated regions, contains a predictedmethionine-initiated coding sequence of about 1166 nucleotides(nucleotides 220-1386 of SEQ ID NO:9). The coding sequence encodes a 388amino acid protein (SEQ ID NO:10).

The human DGAT2 family member sequence 60489 (SEQ ID NO:7), which isapproximately 1255 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 1025nucleotides (nucleotides 170-1195 of SEQ ID NO:7) The coding sequenceencodes a 341 amino acid protein (SEQ ID NO:8).

The DGAT2 family member sequence 112041 (SEQ ID NO:19), which isapproximately 1716 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 1013nucleotides (nucleotides 101-1114 of SEQ ID NO:19) The coding sequenceencodes a 337 amino acid protein (SEQ ID NO:20).

The DGAT2 family member sequence 112037 (SEQ ID NO:61), which isapproximately 712 nucleotides long, is a predicted partial codingsequence. The sequence encodes a 236 amino acid protein (SEQ ID NO:62).

The DGAT2 family member sequence of 58765 identified two splice variantsequences including 58765 (SEQ ID NO:1), which is approximately 1005nucleotides long, encodes a 334 amino acid protein (SEQ ID NO:2).Additionally, 58765short (SEQ ID NO:3), which is approximately 855nucleotides long, encodes a 284 amino acid protein (SEQ ID NO:4).

The DGAT2 family member sequence 112023 (SEQ ID NO:13), which isapproximately 1279 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 986nucleotides (nucleotides 42-1028 of SEQ ID NO:13) The coding sequenceencodes a 328 amino acid protein (SEQ ID NO:14).

The DGAT2 family member sequence 112024 (SEQ ID NO:17), which isapproximately 1720 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 1001nucleotides (nucleotides 1-1002 of SEQ ID NO:17) The coding sequenceencodes a 333 amino acid protein (SEQ ID NO:18).

The DGAT2 family member sequence hDC2 (SEQ ID NO:21), which isapproximately 1093 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 1004nucleotides (nucleotides 49-1053 of SEQ ID NO:21) The coding sequenceencodes a 334 amino acid protein (SEQ ID NO:22).

Example 2 Identification and Characterization of Murine DGAT2 FamilyMember cDNAs and Proteins

A number of murine gene sequences were also identified which are relatedto DGAT2 sequences. The murine DGAT2 sequence (m86606) is depicted inSEQ ID NO:11, which is approximately 2262 nucleotides long includinguntranslated regions, contains a predicted methionine-initiated codingsequence of about 1166 nucleotides (nucleotides 207-1373 of SEQ IDNO:11). The coding sequence encodes a 388 amino acid protein (SEQ IDNO:12).

The murine DGAT2 family member sequence m58765 sequence (SEQ ID NO:5),which is approximately 1748 nucleotides long including untranslatedregions, contains a predicted methionine-initiated coding sequence ofabout 758 nucleotides (nucleotides 254-1012 of SEQ ID NO:5). The codingsequence encodes a 252 amino acid protein (SEQ ID NO:6).

The DGAT2 family member sequence ml 12023 (SEQ ID NO:15), which isapproximately 1255 nucleotides long including untranslated regions,contains a predicted methionine-initiated coding sequence of about 1124nucleotides (nucleotides 27-1151 of SEQ ID NO:15) The coding sequenceencodes a 374 amino acid protein (SEQ ID NO:16).

The DGAT2 family member cDNA sequence mDC2 (SEQ ID NO:23), which isapproximately 1008 nucleotides encodes a 335 amino acid protein (SEQ IDNO:24).

Example 3 DGAT2 Family Member Gene Expression in Human and Mouse TissuesRNA Samples

Human tissue samples were either purchased from Invitrogen or wereprepared from samples available at Millennium. Total RNA samples fromvarious mouse tissues were extracted from 8 week old female mice. Allmice were purchased from Jackson Labs. To investigate tissuedistribution of these genes, cDNAs were prepared from RNA samples priorto Taqman analysis.

RNA was prepared using the trizol method and treated with DNAse toremove contaminating genomic DNA. cDNA was synthesized using randomhexamer primers. Mock cDNA synthesis in the absence of reversetranscriptase resulted in samples with no detectable PCR amplificationof the control 18S gene confirming efficient removal of genomic DNAcontamination. Taqman analysis was performed following themanufacturer's directions.

PCR probes were designed by PrimerExpress software (PE Biosystems) basedon the respective sequences of murine and human genes. The followingprobes and primers were used:

86606 forward primer: (SEQ ID NO: 25) CAAGCCCCTTTATTGCCACTAC 86606reverse primer: (SEQ ID NO: 26) TCCCCTTGGCAGAGAAACTG 86606 Probe: (SEQID NO: 27) CCACGCTCGTCTAGTCCTGAAACTGCAG m86606 forward primer: (SEQ IDNO: 28) TTCCCCAGACGACAGACACTT m86606 reverse primer: (SEQ ID NO: 29)CTCTCAAGAATCCCTGGAGTCACT m86606 Probe: (SEQ ID NO: 30)ACTGCCCTTGCCCAGCTAGCCAGTACTGCCCTTGCC CAGCTAGCCAG hDC2 forward primer:(SEQ ID NO: 31) CTATAGGAAAGCCATCCACACTGTT hDC2 reverse primer: (SEQ IDNO: 32) GGGTCGGGTTCAGAGTCTGA hDC2Probe: (SEQ ID NO: 33)TTGGCCGCCCGATCCCTGT mDC2 forward primer: (SEQ ID NO: 34)GGCTCACCCAGGAACATTCA mDC2 reverse primer: (SEQ ID NO: 35)GGTCAAGGCCATCTTAACAAACC mDC2 Probe: (SEQ ID NO: 36) CTGTGCATCCGCCAGCGCAA112023 forward primer: (SEQ ID NO: 37) GCGGCCACAAGGATGTAAA 112023reverse primer: (SEQ ID NO: 38) GAGCTACCTTGCCATCTTTTGG 112023 Probe:(SEQ ID NO: 39) AGCAGGTAGACGAACAATGGCTGCAAGATCTTGCAGCCATTGTTCGTCTACCTGCT m112023 forward primer: (SEQ ID NO: 40)CGTTGCCATGTTTTGGATTG m112023 reverse primer: (SEQ ID NO: 41)TGTTGGTAGCGGCCACAA m112023 Probe: (SEQ ID NO: 42)CAGCCATTGTTAATTTGCCTATTGTTCACACC 112024 forward primer: (SEQ ID NO: 43)TCAATGCTGGCACCAAAGTG 112024 reverse primer: (SEQ ID NO: 44)TGGTGAGATAGTCCCAAGAAACAG 112024 Probe: (SEQ ID NO: 45)AGGCCCGTCTCCCCTAGGCTCTTC m58765 forward primer: (SEQ ID NO: 46)GGTGAGTGCCGATCACATTCT m58765 reverse primer: (SEQ ID NO: 47)CAACGATGATGGCAAGCAAGT m58765 Probe: (SEQ ID NO: 48)TCCAGGAAGGGCGGCGGGCCCGCCGCCCTTCCTGGA 58765 forward primer: (SEQ ID NO:49) TGACCGCGCCATTTCCTA 58765 reverse primer: (SEQ ID NO: 50)GATTCAGACTGGTCCAAACCCTAT 58765 Probe: (SEQ ID NO: 51)TCCTTCCATGACCCTCCATTGCTCCTAG 58765s forward primer: (SEQ ID NO: 52)CCTGGATCCTTCACGCTGTTAC 58765s reverse primer: (SEQ ID NO: 53)AGGCTTGATACCCGTGTGTCA 58765s Probe: (SEQ ID NO: 54)CGGAACCGAAAGGGCTTCGTCAGCTGACGAAGCCC TTTCGGTTCCG 60489 forward primer:(SEQ ID NO: 55) CGAGGAGGAAGTCAATCACTATCA 60489 reverse primer: (SEQ IDNO: 56) TTTCCTTGTGCTCCTCGAAGA 60489 Probe: (SEQ ID NO: 57)CCCTCTACATGACGGACCTGGAGCAG 112041 forward primer: (SEQ ID NO: 58)GAGACCCAAGAGCTGACAATTACA 112041 reverse primer: (SEQ ID NO: 59)TGGATCCCTCATGGCTTTG 112041 Probe: (SEQ ID NO: 60)AACAGGAGCCACATTCCCCATTGATCA 112037 forward primer: (SEQ ID NO: 63)CCTGCCTCTTCCCCAAACTC 112037 reverse primer: (SEQ ID NO: 64)GAAGAAGAGGAGATGGAACCAACA 112037 probe: (SEQ ID NO: 65)CGCCACACCTGCTCATGCTGC

To allow standardization between different tissues, each samplecontained two probes distinguished by different fluorescent labels, aprobe for the gene of interest (e.g. 86606) as well as a probe for 18SRNA as an internal control. The threshold values at which the PCRamplification started were determined using the manufacturer's software.

The following method was used to quantitatively calculate geneexpression in the tissue samples, relative to the 18S RNA expression inthe same tissue. The threshold values at which the PCR amplificationstarted were determined using the manufacturer's software. PCR cyclenumber at threshold value was designated as CT. Relative expression wascalculated as2^(−((CTtest-CT18S) tissue of interest−(CTtest−CT18S) lowest expressing tissue in panel)).Samples were run in duplicate and the averages of 2 relative expressionlevels that were linear to the amount of template cDNA with a slopesimilar to the slope for the internal control 18S were used. Theresulting relative expression levels for each gene of interest werecompiled and calculated in separate experiments.

TABLE 2 DGAT2 family member expression in human tissues tissue 86606hDC2 112024 58765 58765 s 60489 112041 112037 adipose 1005 2642 2.124347.0 183.7 136.5 404.8 6.801 brain 24.86 1146 1.218 8.503 28.45 6.013235.7 3.866 heart 19.90 1389 1.266 23.37 8.462 3.451 103.4 1.431 kidney13.38 8975 0.918 3399 1660 9950 214.6 5.684 liver 349.8 13827 1.30730902 19076 2445 56.00 3.599 pancreas 1.014 125.4 1.219 9.563 2.6291.091 31.04 2.464 spleen 9.242 1.086 1.390 39.34 18.71 6.291 141.1 4.114s. intestine 22.99 48.77 2.045 1773348 60400 65083 685.2 56.54 sk.muscle 1.894 104.06 1.124 8.800 7.425 2.836 5.257 1.838

The results of expression of 86606 in human tissues by Taqman analysisshowed highest levels of expression in adipose and medium level in liverand lower levels in brain, heart, kidney and small intestine, among thenine human tissues that we have investigated. hDC2 is expressed athighest levels in liver and kidney, and at a lower level in adipose,brain and heart in human tissues tested. The expression of 112024 isvery low in all the human tissues that we examined. 58765 has twosplicing variants, the short form (58765short) lacks part of theC-terminus compared with the long form (58765). Both forms of 58765 arehighly expressed in small intestine, as well as the liver, and at lowerlevels in kidney and adipose tissue. 60489 is expressed highly in smallintestine as well as the kidney, and at lower levels in liver andadipose tissues. 112041 is expressed at higher levels in small intestineand adipose tissues compared with other tissues that we haveinvestigated in human. 112037 is expressed in the small intestine, andat lower levels in adipose and kidney.

In addition to the initial nine human tissues tested, we examinedexpression of 58765short and 60489 in an additional panel of humantissues (Table 3). 58765short and 60489 demonstrated highest expressionin small intestine (as seen in Table 2 above), as well as significantexpression in colon, with lower expression in liver which is upregulatedin liver fibrosis (Table 3). Tissues also tested which did notdemonstrate significant expression levels include erythroid,megakaryocytes, neutrophils, activated PBMCs, hematopoietic progenitorcells (erythroid, megakaryocyte, neutrophil), synovium, macrophages,lymph node, spleen, lung (normal, COPD, and tumor), prostate (normal andtumor), breast (normal and tumor), ovary tumor, dorsal root ganglion,pancreas, nerve, hypothalamus, pituitary gland, brain cortex, spinalcord, skin, adrenal cortex, bladder, primary osteoblast, adipose,skeletal muscle, heart (normal and CHF), hemangioma, HUVEC, coronarySMC, and vessel (artery, vein, and diseased aorta) tissue.

TaqMan analysis was also performed in mouse tissues as indicated above.The mouse orthologue of 86606, m86606 is expressed highly in both whiteand brown adipose tissues in mouse, with lower levels of expression inliver, heart, small intestine and kidney; mDC2 is expressed at highestlevels in both brown and white adipose tissues as well as kidney inmouse; ml 12023 is low in all tissues that we examined. Among thesetissues, the relative expression level is lung>spleen>w fat, b fat>othertissues; and m58765, similar to human 58765, is highly expressed insmall intestine, with lower levels of expression in kidney and adiposetissue in mouse (Table 4).

TABLE 3 DGAT2 family member expression in human tissues Tissue Type58765short 60489 Kidney 0.086 0.2681 Small intestine normal 4.17217.2641 Ovary normal 0.3739 1.6198 Colon normal 2.4466 6.1936 Colon Tumor1.6827 6.8248 Colon IBD 0.2375 3.14 Liver normal 0.674 0.9868 Liverfibrosis 1.5919 4.3493 Tonsil normal 0.0309 0

TABLE 4 DGAT2 family member expression in mouse tissues tissue m86606mDC2 m112023 m58765 brain 16.9188 4.9502 1.1598 5.0109 hypothalamus9.71379 18.1208 1.4636 — heart 78.0162 0.5721 1.4056 233.2449 kidney32.197 304.62 1.1728 588.3001 liver 89.0194 3.8243 1.2581 4.18738 lung11.1751 9.003 111.3496 7.8561 spleen 1.07834 4.2266 26.6539 3.0421 s.intestine 60.3415 1.3351 5.7179 10903.28 muscle 9.37499 2.8787 1.148733.5309 adipose — — — 0.9569 w fat 943.759 207.577 6.0579 242.4002 b fat537.813 189.39 1.3619 137.4226

In addition to the initial nine murine tissues tested, we examinedexpression of m58765 in an additional panel of mouse tissues (Table 5).m58765 demonstrated highest expression in intestine and kidney (as seenin Table 4 above), (Table 5). Tissues also tested which did notdemonstrate significant expression levels (0.0001 or below) includeSalivary Gland/Normal/MPI197, Hypothalamus/Normal/MET237, SpinalCord/Normal/MET238, Lung/Normal/MET148, Esophagus/Normal/MET143,Liver/Normal/MP1149, Brain/Normal/MPI195, Skin/Normal/MET067,Spleen/Normal/MET063, Pancreas/Normal/MET192, PrimaryOsteoblast/Normal/MET198, ST2-0/Normal/MET199, ST2-4/Normal/MET200,Muscle/Normal/MPI1266, and Prostate/Normal/MPI1203

TABLE 5 DGAT2 family member expression in mouse tissues Tissue Typem58765 Intestine/Normal/MET145 0.0749 E10/Normal/MPI1232 0.0164Kidney/Normal/MET146 0.0126 Placenta/Normal/MPI1228 0.0062Heart/Normal/MET142 0.0036 Testes/Normal/MET069 0.0053E17.5/Normal/MPI1020 0.0049 E18.5/Normal/MPI1024 0.0042E19.5/Normal/MPI1067 0.0037 P1.5/Normal/MPI1062 0.0032 E8.5 with yolksac/Normal/MPI1249 0.0058 Diaphysis/Normal/MET202 0.0028Metaphysis/Normal/MET203 0.0021 Brown Fat/Normal/MET138 0.0017E13/Normal/MPI1229 0.0019 E15.5/Normal/MPI1056 0.0019E16.5/Normal/MPI1017 0.0019 E13.5/Normal/MPI1039 0.0017Colon/Normal/MET191 0.0010 Ovary/Normal/MPI1202 0.0009Calveolar/Normal/MET201 0.0009 Uterus/Normal/MET236 0.0008Stomach/Normal/MET160 0.0007 Bladder/Normal/MET139 0.0003 AdrenalGland/Normal/MPI1192 0.0003 Breast/Normal/MPI1226 0.0002Aorta/Normal/MET064 0.0002 White Fat/Normal/MET162 0.0002

Examples 4-7 Regulation of DGAT2 Family Member Expression

To determine whether DGAT2 family member expression is regulated underconditions that affect adipocyte differentiation or white adipocytemetabolism, expression of DGAT2 family member was measured in cells ortissues of mice exposed to various conditions. For analyses, TaqMananalysis was performed as indicated above.

Example 4 Regulation of DGAT2 Family Members During AdipocyteDifferentiation DGAT2 Family Member Expression During 3T3-F442ADifferentiation

We tested expression of m86606 during differentiation of thepreadipocyte cell line 3T3-F442A. 3T3-F442A preadipocytes were grown inDMEM containing 10% Calf Serum. Once they reached confluency (designedas day 0), they were induced to differentiate by culturing in DMEMcontaining 10 μg/ml insulin, 0.5 mM isobutyl-methylxanthine, 1 μMDexamethasone and 10% FBS in DMEM. Forty-eight hours post-induction,cells were maintained in 10% FBS in DMEM with 2.5 μg/ml insulin. Mediumwas replaced every two days. Cells were harvested at day 0 and day 10post-induction of differentiation. Total RNA was extracted and cDNAswere made from these samples and subjected to Taqman analysis.

m86606 was expressed at very low levels in preadipocytes and wasdramatically upregulated during adipocyte differentiation, consistentwith expression of 86606 in adipocytes rather than other cell types inthe adipose tissue (Table 6).

TABLE 6 DGAT2 family member expression during 3T3-F442A differentiationDAY m86606 0 1.0434 13 139.5925

DGAT2 Family Member Expression in Human Preadipocytes and PrimaryAdipocytes

Expression of 86606 and hDC2 were assessed in human preadipocytes anddifferentiated human adipocytes. Total RNAs of human primary adipocytes(HPA) and human subcutaneous preadipocytes (HSPA) were purchased fromZen-Bio, Inc. cDNAs were made from these samples and subjected to Taqmananalysis. 86606 was expressed at very low levels in preadipocytes anddramatically upregulated during the differentiation of human primaryadipocytes. hDC2 was expressed at similar levels in both pre- andprimary adipocytes and did not demonstrate upregulation upondifferentiation (Table 7).

TABLE 7 DGAT2 family member expression in human preadipocytes andadipocytes tissue 86606 hDC2 HPA 1792.1694 142.5415 HSPA 3.9581 234.6578

Example 5 Regulation of DGAT2 Family Member in Diet Induced Obese Mice

To examine the regulation of these genes in a diet induced obesity mousemodel, 6 week old C57 BL/6 male mice were fed with either a high-fatdiet or a chow-diet for 24 weeks. White adipose tissues were collectedfrom these mice for Taqman analysis. m86606 and mDC2 mRNA are downregulated in WAT from mice fed with a high fat (HF)-diet compared withmice fed with a chow-diet (Table 8).

TABLE 8 DGAT2 family member expression in WAT from mice fed varied dietsDiet m86606 mDC2 High Fat 1.0664 1.0396 Chow 1.627 2.3457

Example 6 Regulation of DGAT2 Family Members in Genetically Obese Mice

To investigate the regulation of these genes in genetic obese mousemodel, white adipose tissue were collected from male, 8 week old ob/obmice and their lean littermates for Taqman analysis. White adiposetissues were collected from these mice for Taqman analysis. mDC2expression was considerably lower in ob/ob mice compared to wild-typecontrol mice (Table 9). m86606 expression did not change considerably inob/ob mice when compared to wild-type control mice.

TABLE 9 DGAT2 family member expression in WAT from ob/ob and WT micegenotype mDC2 m86606 WT 9132.42 319.58 ob/ob 407.32 280.46

Example 7 Regulation of DGAT2 Family Member During Fasting and Refeeding

Stimulation of lipolysis is believed to be an effective strategy fordecreasing body weight. To examine a possible role of DGAT2 familymember in lipolysis we examined its expression in white adipose tissuesof mice which had been fasted for 3 days. Under those conditions,lipolysis is maximally stimulated and mice rely on fatty acids releasedfrom adipose tissue as an energy source. Fasting mice for 3 daysdecreased m86606 and mDC2 expression in white adipose tissue. Refeedingfor 1 and 2 days caused an increase compared to fasted animals (Table10).

TABLE 10 DGAT2 family member expression in WAT during fasting andrefeeding treatment m86606 mDC2 control 6.065 3.9457 3 d starvation1.0625 1.0396 1 d refeeding 16.1175 3.5233 2 d refeeding 18.1614 4.0593

Example 8 Recombinant Expression of DGAT2 Family Members in BacterialCells

For expression of recombinant DGAT2 family member, aglutathione-S-transferase (GST) fusion polypeptide of a DGAT2 familymember protein is expressed in E. coli, isolated and characterized.Specifically, a DGAT2 family member polypeptide is genetically fused toGST and this fusion polypeptide is expressed in E. Coli, e.g., strainPEB199. Expression of the GST-DGAT2 family member fusion protein inPEB199 is induced with IPTG. The recombinant fusion polypeptide ispurified from crude bacterial lysates of the induced PEB199 strain byaffinity chromatography on glutathione beads. Using polyacrylamide gelelectrophoretic analysis of the polypeptide purified from the bacteriallysates, the molecular weight of the resultant fusion polypeptide isdetermined.

Example 9 Expression of Recombinant DGAT2 Family Members Protein inMammalian Cells

To express a DGAT2 family member gene in mammalian cells, for exampleCOS cells, the pcDNA/Amp vector by Invitrogen Corporation (San Diego,Calif.) is used. This vector contains an SV40 origin of replication, anampicillin resistance gene, an E. coli replication origin, a CMVpromoter followed by a polylinker region, and an SV40 intron andpolyadenylation site. A DNA fragment encoding the entire DGAT2 familymember protein of interest and an HA tag (Wilson et al. (1984) Cell37:767) or a FLAG tag fused in-frame to its 3′ end of the fragment iscloned into the polylinker region of the vector, thereby placing theexpression of the recombinant protein under the control of the CMVpromoter.

To construct the plasmid, the DGAT2 family member DNA sequence isamplified by PCR using two primers. The 5′ primer contains therestriction site of interest followed by approximately twentynucleotides of the DGAT2 family member coding sequence starting from theinitiation codon; the 3′ end sequence contains complementary sequencesto the other restriction site of interest, a translation stop codon, theHA tag or FLAG tag and the last 20 nucleotides of the DGAT2 familymember coding sequence. The PCR amplified fragment and the pCDNA/Ampvector are digested with the appropriate restriction enzymes and thevector is dephosphorylated using the CIAP enzyme (New England Biolabs,Beverly, Mass.). Preferably the two restriction sites chosen aredifferent so that the DGAT2 family member gene is inserted in thecorrect orientation. The ligation mixture is transformed into E. colicells (strains HB101, DH5α, SURE, available from Stratagene CloningSystems, La Jolla, Calif., can be used), the transformed culture isplated on ampicillin media plates, and resistant colonies are selected.Plasmid DNA is isolated from transformants and examined by restrictionanalysis for the presence of the correct fragment.

COS cells are subsequently transfected with the DGAT2 familymember-pcDNA/Amp plasmid DNA using the calcium phosphate or calciumchloride co-precipitation methods, DEAE-dextran-mediated transfection,lipofection, or electroporation. Other suitable methods for transfectinghost cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T.Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring HarborLaboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y., 1989. The expression of the DGAT2 family member polypeptide isdetected by radiolabelling (³⁵S-methionine or ³⁵S-cysteine availablefrom NEN, Boston, Mass., can be used) and immunoprecipitation (Harlow,E. and Lane, D. Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1988) using an HA specificmonoclonal antibody. Briefly, the cells are labeled for 8 hours with³⁵S-methionine (or ³⁵S-cysteine). The culture media are then collectedand the cells are lysed using detergents (RIPA buffer, 150 mM NaCl, 1%NP-40, 0.1% SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate andthe culture media are precipitated with an HA specific monoclonalantibody. Precipitated polypeptides are then analyzed by SDS-PAGE.

Alternatively, DNA containing the DGAT2 family member coding sequence iscloned directly into the polylinker of the pCDNA/Amp vector using theappropriate restriction sites. The resulting plasmid is transfected intoCOS cells in the manner described above, and the expression of the DGAT2family member polypeptide is detected by radiolabelling andimmunoprecipitation using a DGAT2 family member specific monoclonalantibody.

Example 10 Regulation of DGAT2 Family Members During EnterocyteDifferentiation

Caco-2 is a human intestinal cell line. Upon reach confluence, the cellsexpress characteristics of enterocytic differentiation. During Caco-2differentiation, triglyceride synthesis is increased (Pamela J et alJournal of Lipid Research, 1991, 32:293-304). To determine whether theexpression of 58765 and 60489 are also elevated, we examined theexpression of 58765 and 60489 in Caco-2 cells during differentiation.

Caco-2 cells were purchased from ATCC. They were cultured in DMEMcontaining 15% fetal bovine serum. The medium was changed 2-3 times perweek. At day 3, the cells were at subconfluence. They reached confluenceand started differentiating at day 7. At day 25, they were fullydifferentiated. The cells were harvested for RNA extraction at day 3 andday 25 after they were seeded. Taqman analysis was performed asdescribed above to determine relative expression levels of 58765 and60489 (Table 11).

Taqman data demonstrate that both 58765 and 60489 are upregulated duringdifferentiation which correlates well with triglyceride synthesis inthese cells (Table 11). This is consistent with playing a role intriglyceride biosynthesis in the small intestine.

TABLE 10 DGAT2 family member expression during enterocytedifferentiation day 58765 60489  3-1 1.86 1.22  3-2 1.13 1.11 25-1 98.362.79 25-2 91.13 4.02

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. An isolated nucleic acid molecule selected from the group consistingof: a) a nucleic acid molecule comprising a nucleotide sequence which isat least 85% identical to the nucleotide sequence of SEQ ID NO:7, SEQ IDNO:19, or SEQ ID NO:61; b) a nucleic acid molecule comprising a fragmentof at least 300 nucleotides of the nucleotide sequence of SEQ ID NO:7,SEQ ID NO:19 or SEQ ID NO:61; c) a nucleic acid molecule which encodes apolypeptide comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:12, SEQ ID NO:14, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20 and SEQ IDNO:62; d) a nucleic acid molecule which encodes a fragment of apolypeptide comprising the amino acid sequence of SEQ ID NO:8, SEQ IDNO:20, or SEQ ID NO:62, wherein the fragment comprises at least 15contiguous amino acids of SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:62; e)a nucleic acid molecule which encodes a naturally occurring allelicvariant of a polypeptide comprising the amino acid sequence of SEQ IDNO:8, SEQ ID NO:20 or SEQ ID NO:62, wherein the nucleic acid moleculehybridizes to a nucleic acid molecule comprising a sequence consistingof SEQ ID NO:7, SEQ ID NO:19, or SEQ ID NO:61 or a complement thereof,under stringent conditions; and f) a nucleic acid molecule comprising anucleotide sequence selected from the group consisting of SEQ ID NO:1,SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, SEQID NO:17, SEQ ID NO:19, and SEQ ID NO:61.
 2. The nucleic acid moleculeof claim 1 further comprising vector nucleic acid sequences or nucleicacid sequences encoding a heterologous polypeptide.
 3. A host cell whichcontains the nucleic acid molecule of claim
 1. 4. An isolatedpolypeptide selected from the group consisting of: a) a polypeptidewhich is encoded by a nucleic acid molecule comprising a nucleotidesequence which is at least 85% identical to a nucleic acid comprisingthe nucleotide sequence of SEQ ID NO:7, SEQ ID NO:19 or SEQ ID NO:61, ora complement thereof; b) a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:8, SEQ IDNO:20 or SEQ ID NO:62, wherein the polypeptide is encoded by a nucleicacid molecule which hybridizes to a nucleic acid molecule comprising SEQID NO:7, SEQ ID NO:19 or SEQ ID NO:61 or a complement thereof understringent conditions; c) a fragment of a polypeptide comprising theamino acid sequence of SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:62,wherein the fragment comprises at least 15 contiguous amino acids of SEQID NO:8, SEQ ID NO:20 or SEQ ID NO:62; and d) a polypeptide comprisingthe amino acid sequence selected from the group consisting of SEQ IDNO:2, SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:18, SEQ ID NO:20 and SEQ ID NO:62.
 5. Thepolypeptide of claim 4 further comprising heterologous amino acidsequences.
 6. An antibody which selectively binds to a polypeptide ofclaim
 4. 7. A method for producing a polypeptide selected from the groupconsisting of: a) a polypeptide comprising the amino acid sequenceselected from the group consisting of SEQ ID NO:2, SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:15, SEQ IDNO:18, SEQ ID NO:20, and SEQ ID NO:62; b) a polypeptide comprising afragment of the amino acid sequence of SEQ ID NO:8 or SEQ ID NO:20,wherein the fragment comprises at least 15 contiguous amino acids of SEQID NO:8, SEQ ID NO:20, or SEQ ID NO:62; c) a polypeptide which isencoded by a nucleic acid molecule comprising a nucleotide sequencewhich is at least 85% identical to a nucleic acid comprising thenucleotide sequence of SEQ ID NO:7, SEQ ID NO:19, or SEQ ID NO:61, or acomplement thereof; and d) a naturally occurring allelic variant of apolypeptide comprising the amino acid sequence of SEQ ID NO:8, SEQ IDNO:20 or SEQ ID NO:62, wherein the polypeptide is encoded by a nucleicacid molecule which hybridizes to a nucleic acid molecule comprising SEQID NO:7, SEQ ID NO:19, SEQ ID NO:61, or a complement thereof understringent conditions; comprising culturing the host cell of claim 3under conditions in which the nucleic acid molecule is expressed.
 8. Amethod for identifying a compound which binds to a polypeptide of claim4 comprising the steps of: a) contacting a polypeptide, or a cellexpressing a polypeptide of claim 4 with a test compound; and b)determining whether the polypeptide binds to the test compound; whereinbinding of the test compound to the polypeptide is detected by a methodselected from the group consisting of: i) detection of binding by directdetecting of test compound/polypeptide binding; ii) detection of bindingusing a competition binding assay; and iii) detection of binding usingan assay for diacylglycerol acyltransferase activity.
 9. A method foridentifying a compound which modulates the activity of a polypeptide ofclaim 4, comprising: a) contacting a polypeptide of claim 4 with a testcompound; and b) determining the effect of the test compound on thediacylglycerol acyltransferase activity of the polypeptide to therebyidentify a compound which modulates the activity of the polypeptide. 10.A method for identifying a compound capable of treating a disordercomprising: a) contacting an adipocyte with a test compound; and b)assaying the ability of the test compound to modulate the expression ofa DGAT2 family member nucleic acid or the activity of a DGAT2 familymember polypeptide; and c) identifying a compound capable of modulatingan adipocyte activity when a test compound modulates the expression of aDGAT2 family member nucleic acid or the activity of a DGAT2 familymember polypeptide.
 11. The method of claim 10, wherein said adipocyteactivity comprises any one of DGAT2 family member nucleic acidexpression, diacylglyceroltransferase activity, hyperplastic growth,hypertrophic growth, or lipogenesis.
 12. A method of identifying acompound which modulates the activity of a polypeptide of claim 4,comprising: a) combining a sample comprising the polypeptide with afirst fatty acyl coA substrate and a second acylglyceride substrateunder conditions suitable for enzyme activity; b) contacting the samplecomprising the acyltransferase and substrates with a test compound; c)determining the amount of acylglycerol product formation, therebydetermining acyltransferase activity; and d) comparing theacyltransferase activity in the presence of compound withacyltransferase activity in the absence of compound; wherein adifference between the acyltransferase activity in the presence ofcompound with acyltransferase activity in the absence of compoundindicates that the compound modulates the activity of the polypeptide,thereby identifying a compound which modulates activity of theacyltransferase.
 13. The method of claim 12, wherein the method detectsdiacylglycerol acyltransferase activity or monoacylglycerolacyltransferase activity.
 14. The method of claim 12, wherein onesubstrate is biotinylated and the other substrate is radiolabeled.
 15. Amethod for treating a subject having an obesity disorder characterizedby expression or activity of a polypeptide of claim 4, comprisingadministering to the subject a DGAT2 family member modulator, therebytreating said subject having an obesity disorder.
 16. A method fordetecting the presence of a nucleic acid molecule of claim 1 in asample, comprising: a) contacting the sample with a compound whichselectively hybridizes to a nucleic acid molecule of claim 1; and b)determining whether the compound hybridizes to the nucleic acid or bindsto the polypeptide in the sample.
 17. A method for detecting thepresence of a polypeptide of claim 4 in a sample, comprising: a)contacting the sample with a compound which binds to the polypeptide;and b) determining whether the compound hybridizes to the nucleic acidor binds to the polypeptide in the sample.
 18. A method of identifying asubject having a disorder or at risk for developing a disordercomprising: a) contacting a sample obtained from the subject comprisingnucleic acid molecules with a hybridization probe comprising at least 25contiguous nucleotides of a nucleic acid molecule of claim 1; and b)detecting the presence of a nucleic acid molecule in the sample thathybridizes to the probe, thereby identifying a subject having a disorderor at risk for developing a disorder.
 19. A method of identifying asubject having a disorder or at risk for developing a disordercomprising: a) contacting a sample obtained from the subject comprisingpolypeptides with a compound which binds a polypeptide of claim 4; andb) detecting the presence of a polypeptide in the sample that binds tothe compound, thereby identifying a subject having a disorder or at riskfor developing a disorder.